N-sulfonylarginine keto-amide compounds

ABSTRACT

The present invention is directed to N-sulfonyl arginine alpha-keto-amide derivatives, their pharmaceutically acceptable salts and compositions thereof which are useful as antithrombotic agents in mammals and also the use of these compounds as antithrombotic agents. Also, disclosed are methods of using these inhibitors in their various embodiments as therapeutic agents for disease states characterized by disorders of the blood coagulation process. Further disclosed are compounds useful as intermediates in the preparation of these compounds.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/962,301,filed Oct. 16, 1992, entitled "Arginine Keto-Amide Enzyme Inhibitors",now U.S. Pat. No. 5,371,072, hereby incorporated by reference herein,including the drawings attached thereto.

FIELD OF THE INVENTION

The present invention relates in one aspect to novel compounds, theirpharmaceutically acceptable salts and pharmaceutically acceptablecompositions thereof which are useful as potent and specific inhibitorsof blood coagulation in mammals. In another aspect, the inventionrelates to a methods of using certain of these inhibitors as therapeuticagents for disease states characterized by disorders of the bloodcoagulation process. In yet another aspect, the invention relates tointermediate compounds for the preparation of the inhibitors.

BACKGROUND AND INTRODUCTION TO THE INVENTION

Normal hemostasis is the result of a complex balance between theprocesses of clot formation (blood coagulation) and clot dissolution(fibrinolysis). The complex interactions between blood cells, specificplasma proteins and the vascular surface, maintain the fluidity of bloodunless injury and blood loss occur.

Blood coagulation is the culmination of a series of amplified reactionsin which several specific zymogens of serine proteases in plasma areactivated by limited proteolysis. Nemerson, Y. and Nossel, H. L., Ann.Rev. Med., 33: 479 (1982). This series of reactions results in theformation of an insoluble fibrin matrix which is required for thestabilization of the primary hemostatic plug. The interaction andpropagation of the activation reactions occurs through the extrinsic andintrinsic pathways of coagulation. These pathways are highlyinterdependent and converge in the formation of the serine protease,Factor Xa. Factor Xa catalyzes the penultimate step in the bloodcoagulation cascade which is the formation of the serine proteasethrombin. This step occurs following the assembly of the prothrombinasecomplex which is composed of factor Xa, the non-enzymatic co-factor Vaand the substrate prothrombin assembled on the surface of adhered,activated platelets or systemically circulating membranousmicroparticles.

Proteolytic activation of zymogen factor X to its catalytically activeform, factor Xa, can occur by either the intrinsic or extrinsiccoagulation pathways. The intrinsic pathway is referred to as"intrinsic" because everything needed for clotting is in the blood.Saito, H., "Normal Hemostatic Mechanisms", Disorders of Hemostasis, pp.27-29, Grune & Stratton, Inc. (O. D. Ratnoff, M.D. and C. D. Forbes,M.D. edit. 1984). This pathway is comprised of the zymogen serineproteases, factors IX and XI, and the non-enzymatic co-factor, factorVIII. The initiation of the intrinsic pathway results in the activationof factor XI to XIa. Factor XIa catalyzes the activation of factor IX tofactor IXa which in combination with the activated form of factor VIIIon an appropriate phospholipid surface, results in the formation of thetenase complex. This complex also catalyzes the formation of the serineprotease, factor Xa, from its zymogen, factor X which subsequentlyresults in clot formation.

The extrinsic pathway is referred to as "extrinsic" because the tissuefactor which binds to and facilitates the activation of factor VII comesfrom outside the blood. Saito, Id. The major components of this pathwayare the zymogen serine protease, factor VII, and the membrane boundprotein, tissue factor. The latter serves as the requisite non-enzymaticco-factor for this enzyme. The initiation of this pathway is thought tobe an autocatalytic event resulting from the activation of zymogenfactor VII by trace levels of activated factor VII (factor VIIa), bothof which are bound to newly exposed tissue factor on membrane surfacesat sites of vascular damage. The factor VIIa/tissue factor complexdirectly catalyzes the formation of the serine protease, factor Xa, fromits zymogen, factor X. Exposure of blood to injured tissue initiatesblood clotting by the extrinsic pathway.

Proteolytic activation of zymogen factor X to its catalytically activeform, factor Xa, results in the liberation of a 52 amino acid activationpeptide from the amino-terminus of the heavy chain subunit. Theintrinsic activation reaction is catalyzed by factor IXa in amacromolecular complex with the non-enzymatic co-factor, factor VIIIa.Factor Xa formation via the extrinsic pathway is catalyzed by thecatalytic complex of factor VIIa and tissue factor. Both of thesereactions must occur on an appropriate phospholipid surface in thepresence of calcium ions. The active product formed following eitherintrinsic or extrinsic activation of factor X is α-factor Xa. A secondproteolytic cleavage which is thought to be autocatalytic, results inthe formation of β-factor Xa following the release of a 14 amino acidpeptide from the carboxy-terminus of the heavy chain. Both forms of theactivated molecule have the same catalytic activity as measured by theirability to promote coagulation in plasma or hydrolyze a peptidylchromogenic substrate.

The formation of thrombin is catalyzed by factor Xa following theassembly of the catalytic prothrombinase complex as reviewed by Mann, K.G. et. al., "Surface-Dependent Reactions of the Vitamin K-DependentEnzyme Complexes", Blood, 76: 1-16 (1990). This complex is composed offactor Xa, the non-enzymatic co-factor Va and the substrate prothrombinall assembled on an appropriate phospholipid surface. The requirement ofa macromolecular complex for efficient catalysis results in theprotection of factor Xa from natural anticoagulant mechanisms such asheparin-antithrombin III mediated inhibition. Teite, J. M. andRosenberg, R. D., "Protection of Factor Xa from neutralization by theheparin-antithrombin complex", J. Clin. Invest., 71: 1383-1391 (1983).In addition, sequestration of factor Xa in the prothrombinase complexalso renders it resistant to inhibition by exogenous heparin therapywhich also requires antithrombin III to elicit its anticoagulant effect.

Thrombin is the primary mediator of thrombus formation. Thrombin actsdirectly to cause formation of insoluble fibrin from circulatingfibrinogen. In addition, thrombin activates the zymogen factor XIII tothe active transglutaminase factor XIIIa which acts to covalentlystabilize the growing thrombus by crosslinking the fibrin strands.Lorand, L. and Konishi, K., Arch. Biochem. Biophys., 105: 58 (1964).Beyond its direct role in the formation and stabilization of fibrin richclots, the enzyme has been reported to have profound bioregulatoryeffects on a number of cellular components within the vasculature andblood. Shuman, M. A., Ann. NY Acad. Sci., 405: 349 (1986).

It is believed that thrombin is the most potent agonist of plateletactivation, and it has been demonstrated to be the primarypathophysiologic-mediator of platelet-dependent arterial thrombusformation. Edit, J. F. et al., J. Clin. Invest., 84: 18 (1989).Thrombin-mediated platelet activation leads to ligand-inducedinter-platelet aggregation principally due to the bivalent interactionsbetween adhesive ligands such as fibrinogen and fibronectin withplatelet integrin receptors such as glycoprotein IIb/IIIa which assumetheir active conformation following thrombin activation. Berndt, M. C.,and Phillips, D. R., Platelets in Biology and Pathology, pp 43-74,Elsevier/North Holland Biomedical Press (Gordon, J. L. edit. 1981).Thrombin-activated platelets can also support further thrombinproduction through the assembly of new prothrombinase and tenase (factorIXa, factor VIIIa and factor X) catalytic complexes on the membranesurface of intact activated platelets and platelet-derivedmicroparticles, following thrombin-mediated activation of thenon-enzymatic cofactors V and VIII, respectively. Tans, G. et al.,Blood, 77: 2641 (1991). This positive feedback process results in thelocal generation of large concentrations of thrombin within the vicinityof the thrombus which supports further thrombus growth and extension.Mann, K. G. et al., Blood, 76: 1 (1990).

In contrast to its prothrombotic effects, thrombin has been shown toinfluence other aspects of hemostasis. These include its effect as animportant physiological anticoagulant. The anticoagulant effect ofthrombin is expressed following binding of thrombin to the endothelialcell membrane glycoprotein, thrombomodulin. This is thought to result inan alteration of the substrate specificity of thrombin thereby allowingit to recognize and proteolytically activate circulating protein C togive activated protein C (aPC). Musci, G. et al., Biochemistry, 27: 769(1988). aPC is a serine protease which selectively inactivates thenon-enzymatic co-factors Va and VIIIa resulting in a down-regulation ofthrombin formation by the prothrombinase and tenase catalytic complexes,respectively. Esmon, C. T., Science, 235: 1348 (1987). The activation ofprotein C by thrombin in the absence of thrombomodulin is poor.

Thrombin has also been shown to be a potent direct mitogen for a numberof cell types, including cells of mesenchymal origin such as vascularsmooth muscle cells. Chen, L. B. and Buchanan, J. M., Proc. Natl. Acad.Sci. USA, 72: 131 (1975). The direct interaction of thrombin withvascular smooth muscle also results in vasoconstriction. Walz, D. A. etal., Proc. Soc. Expl. Biol. Med., 180: 518 (1985). Thrombin acts as adirect secretagogue inducing the release of a number of bioactivesubstances from vascular endothelial cells including tissue plasminogenactivator. Levin, E. G. et al., Thromb. Haemost., 56: 115 (1986). Inaddition to these direct effects on vascular cells, the enzyme canindirectly elaborate potent mitogenic activity on vascular smooth musclecells by the release of several potent growth factors (e.g.platelet-derived growth factor and epidermal growth factor) fromplatelet α-granules following thrombin-induced activation. Ross, R., N.Engl. J. Med., 314: 408 (1986).

Many significant disease states are related to abnormal hemostasis. Withrespect to the coronary arterial vasculature, abnormal thrombusformation due to the rupture of an established atherosclerotic plaque isthe major cause of acute myocardial infarction and unstable angina.Moreover, treatment of an occlusive coronary thrombus by eitherthrombolytic therapy or percutaneous transluminal coronary angioplasty(PTCA) is often accompanied by an acute thrombotic reclosure of theaffected vessel which requires immediate resolution. With respect to thevenous vasculature, a high percentage of patients undergoing majorsurgery in the lower extremities or the abdominal area suffer fromthrombus formation in the venous vasculature which can result in reducedblood flow to the affected extremity and a predisposition to pulmonaryembolism. Disseminated intravascular coagulopathy commonly occurs withinboth vascular systems during septic shock, certain viral infections andcancer and is characterized by the rapid consumption of coagulationfactors and systemic coagulation which results in the formation oflife-threatening thrombi occurring throughout the vasculature leading towidespread organ failure.

Pathogenic thrombosis in the arterial vasculature is a major clinicalconcern in today's medicine. It is the leading cause of acute myocardialinfarction which is one of the leading causes of death in the westernworld. Recurrent arterial thrombosis also remains one of the leadingcauses of failure following enzymatic or mechanical recanalization ofoccluded coronary vessels using thrombolytic agents or percutaneoustransluminal coronary angioplasty (PTCA), respectively. Ross, A. M.,Thrombosis in Cardiovascular Disorder, p. 327, W. B. Saunders Co.(Fuster, V. and Verstraete, M. edit. 1991); Califf, R. M. and Wilierson,J. T., Id. at p 389. In contrast to thrombotic events in the venousvasculature, arterial thrombosis is the result of a complex interactionbetween fibrin formation resulting from the blood coagulation cascadeand cellular components, particularly platelets, which make up a largepercentage of arterial thrombi. There is currently no effective therapyfor the treatment or prevention of acute arterial thrombosis orrethrombosis since heparin, the most widely used clinical anticoagulantadministered i.v., has not been shown to be universally effective inthis setting. Prins, M. H. and Hirsh, J., J. Am. Coll. Cardiol., 67: 3A(1991).

Besides the unpredictable, recurrent thrombotic reocclusion whichcommonly occurs following PTCA, a profound restenosis of the recanalizedvessel occurs in 30 to 40% of patients 1 to 6 months following thisprocedure. Califf, R. M. et al., J. Am. Coll. Cardiol., 17: 2B (1991).These patients require further treatment with either a repeat PTCA orcoronary artery bypass surgery to relieve the newly formed stenosis.Restenosis of a mechanically damaged vessel is not a thrombotic processbut instead is the result of a hyperproliferative response in thesurrounding smooth muscle cells which over time results in a decreasedluminal diameter of the affected vessel due to increased muscle mass.Id. As for arterial thrombosis, there is currently no effectivepharmacologic treatment for the prevention of vascular restenosisfollowing mechanical recanalization.

The need for safe and effective therapeutic anticoagulants has in oneaspect focused on the role of factor Xa as the catalyst for thepenultimate step in the blood coagulation cascade which is the formationof the serine protease thrombin.

Most preferred natural substrates for thrombin are reported to containan uncharged amino acid in the P3 recognition subsite. For example, thethrombin cleavage site on the Aα chain of fibrinogen, which is theprimary physiological substrate for thrombin, is reported to contain aglycine residue in this position while the cleavage site on the Bβ chaincontains a serine, as shown below:

    ______________________________________    P3 P2 P1 P1'    Gly-Val-Arg/Gly    Fibrinogen Aα Chain    Ser-Ala-Arg/Gly    Fibrinogen Bβ Chain    ______________________________________

Peptidyl derivatives having an uncharged residue in the P3 positionwhich is believed to bind to the active site of thrombin and therebyinhibit the conversion of fibrinogen to fibrin and cellular activationhave been reported. Additionally, these derivatives have either analdehyde, chloromethyl ketone or boronic acid functionality associatedwith the P1 amino acid. For example, substrate-like peptidyl derivativessuch as D-phenylalanyl-prolyl-argininal (D-Phe-Pro-Arg-al),D-phenylalanyl-prolyl-arginine-chloromethyl ketone (P-PACK) andacetyl-D-phenylalanyl-prolyl-boroarginine (Ac-(D-Phe)-Pro-boroArg) havebeen reported to inhibit thrombin by directly binding to the active siteof the enzyme. Bajusz, S., Symposia Biologica Hungarica, 25: 277 (1984),Bajusz, S. et al, J. Med. Chem., 33: 1729 (1990) and Bajusz, S. et al.,Int. J. Peptide Protein Res. 12: 217 (1970); Kettner, C. and Shaw, E.,Methods Enzymol., 80: 826 (1987); Kettner, C. et al., EP 293,881(published Dec. 7, 1988); Kettner, C., et al., J. Biol. Chem., 265:18209 (1990). These molecules have been reported to be potentanticoagulants in the prevention of platelet-rich arterial thrombosis.Kelly, A. B. et al., Thromb. Haemostas., 65: 736 at abstract 257 (1991).

Peptidyl compounds which are said to be active site inhibitors ofthrombin but which are said to differ in structure to those containing auncharged amino acid in the P3 recognition subsite have been reported.The compound, Argatroban (also called2R,4R-4-methyl-1-[N-2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-argininal]-2-piperdinecarboxylicacid), is also reported to bind directly to the active site of thrombinand has been thought to be the most potent and selective compound in theclass of non-peptidyl inhibitors of this enzyme. Okamoto, S. et al.,Biochem. Biophys. Res. Commun., 100: 440 (1981). Argatroban has beenreported to be a potent antithrombotic agent in several experimentalmodels of acute arterial thrombosis. Jang, I. K. et al., in bothCirculation, 81: 219 (1990) and Circ. Res., 67: 1552 (1990).

Peptidyl compounds which are said to be inhibitors of thrombin and whosemode of action is thought to be by binding to the active site andanother site on the enzyme have been reported. Hirudin and its variouspeptidyl derivatives have been reported to inhibit both conversion offibrinogen to fibrin and platelet activation by binding to either theactive site and exo site, or exo site only, of thrombin. Markwardt, F.,Thromb. Haemostas., 66: 141 (1991). Hirudin is reported to be a 65 aminoacid polypeptide originally isolated from leech salivary gland extracts.It is said to be one of the most potent inhibitors of thrombin known.Marki, W. E. and Wallis, R. B., Thromb. Haemostas., 64: 344 (1990). Itis reported to inhibit thrombin by binding to both its anion-bindingexo-site and to its catalytic active site which are distinct andphysically distant from each other. Rydel, supra. Hirudin has beenreported to be a potent antithrombotic agent in vitro. Markwardt, F. etal., Pharmazie, 43: 202 (1988); Kelly, A. B. et al., Blood, 77: 1(1991). In addition to its antithrombotic effects, hirudin has beenreported to also effectively inhibit smooth muscle proliferation and theassociated restenosis following mechanical damage to a atheroscleroticrabbit femoral artery. Sarembock, I. J. et al., Circulation, 84: 232(1991).

Hirugen has been reported to be a peptide derived from the anioniccarboxy-terminus of hirudin. It is reported to bind only to the anionbinding exo-site of thrombin and thereby inhibit the formation of fibrinbut not the catalytic turnover of small synthetic substrates which haveaccess to the unblocked active site of the enzyme. Maraganore, J. M. etal., J. Biol. Chem., 264: 8692 (1989); Naski, M. C. et al., J. Biol.Chem., 265: 13484 (1990). The region of hirudin represented by hirugenhas been reported, as according to by x-ray crystallographic analysis,to bind directly to the exo site of thrombin. Skrzypczak-Jankun, E. etal., Thromb. Haemostas., 65: 830 at abstract 507 (1991). Moreover, thebinding of hirugen has also been reported to enhance the catalyticturnover of certain small synthetic substrates by thrombin, indicatingthat a conformational change in the enzyme active site may accompanyoccupancy of the exo-site. Liu, Supra. Hirugen also is reported to blockthrombin-mediated platelet aggregation. Jakubowski, J. A. andMaraganore, J. M., Blood, 75: 399 (1990).

Hirulog has been reported to be a synthetic chimeric molecule comprisedof a hirugen-like sequence linked by a spacer region to the peptide,D-phenylalanyl-prolyl-arginine which is based on a preferred substraterecognition site for thrombin. The hirugen-like sequence is said to belinked to this peptide through the C-terminal end of the peptide.Maraganone, J. M. et al., Biochemistry, 29: 7095 (1990). Hirulog hasbeen reported to be an effective antithrombotic agent in preventing bothfibrin-rich and platelet-rich thrombosis. Maraganone, J. M. et al.,Thromb. Haemostas., 65: 651 at abstract 17 (1991).

Cyclotheonamide A and B, isolated from the marine sponge, Theonella, agenus of marine sponges, have been reported to be inhibitors of thrombinwith an IC₅₀ of 0.076 mg/mL. Structurally, they have been characterizedas cyclic peptides containing an α-keto amide moiety. Fusetani et al.,J. Am. Chem. Soc. 112: 7053-7054 (1991) and Hagihara et al., J. Am.Chem. Soc, 114: 6570-6571 (1992). It has been proposed that the α-ketogroup of the cyclotheonamides may function as an electrophilic mimic ofthe Arg-X scissile amide bond of the thrombin substrates. Hagihara etal., Id, at 6570. The partial synthesis of cyclotheonamide A and thetotal synthesis of cyclotheonamide B have been reported. Wipf et al.,Tetrahedron Lett., 33: 4275-4278 (1992) and Hagihara et al., J. Am.Chem. Soc, 114: 6570-6571 (1992).

α-Keto ester derivatives of N-protected amino acids and peptides havebeen reported as inhibitors of serine proteases, as neutrophil elastaseand cathepsin. G. Mehdi et al., Biochem. Biophys. Res. Commun., 166:595-600 (1990) and Angelastro et al., J. Med. Chem., 33: 11-13 (1990).

Alpha keto-amide derivatives of amino acids and peptides have beenreported to be inhibit proteases. For example, fluoro-substituted ketoamide derivatives have been reported to be inhibitors of proteases.European Patent Application No. 275,101 (published Jul. 20, 1988).L-valyl-L-valyl-3-amino-2-oxovaleryl-D-leucyl-L-valine had been reportedto be an inhibitor of prolyl endopeptidase. Nagai et al., J.Antibiotics, 44: 956-961 (1991). 3-Amino-2-oxo-4-phenylbutanoic acidamide has been reported to be an inhibitor of arginyl aminopeptidase(with inhibitor constant of 1.5 mM), cytosol aminopeptidase (withinhibitor constant of 1.0 mM) and microsomal aminopeptidase (withinhibitor constant of 2.5 mM). Ocain et al., J. Med. Chem., 35: 451-456(1992). 2-Oxo-2-(pyrrolidin-2yl)acetyl derivatives have been reported tobe inhibitors of prolyl endopeptidase. Someno et al., European PatentApplication No. 468,339 (published Jan. 29, 1992). Certain alphaketo-amide derivatives of peptides have been reported to inhibit variousserine and cysteine proteases. Powers J. C., International ApplicationNo. WO 92/12140 (published Jul. 23, 1992).

SUMMARY OF THE INVENTION

The present invention in one aspect is directed to novel compounds whichare useful as in vitro inhibitors of coagulation proteases and as invivo antithrombic agents. These compounds have the structure: ##STR1##wherein (a) A₁ is selected from the group consisting of R₁ --C(O)--, R₁--O--C(O)--, R₁ --NH--C(O)--, R₁ --S(O₂)--, R₁ --O--S(O₂)--, and R₁--NH--S(O₂)--, wherein R₁ is selected from the group consisting of

alkyl of 1 to about 12 carbon atoms,

alkenyl of about 3 to about 6 carbon atoms,

aryl of about 6 to about 14 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂,

aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂,

aralkenyl of about 8 to about 15 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂,

perfluoroalkyl of 1 to about 12 carbon atoms,

perfluoroaryl of about 6 to about 14 carbon atoms,

trimethylsilylalkyl of 4 to about 8 carbon atoms, ##STR2## wherein X₁and X₂ are independently selected from the group consisting of bromo,chloro, fluoro, Y₁ --, HO--, Y₁ --O--, NH₂ --, Y₁ --NH--, (Y₁,Y₂)N--, Y₁--C(O)--NH--, HS--, Y₁ --S--, Y₁ --S(O)--, Y₁ --S(O₂)--, HO--S (O₂)--,Y₁ --O--S(O₂)--, NH₂ --S(O₂)-- and Y₁ --NH--S(O₂)--, wherein Y₁ and Y₂are independently selected from the group consisting of trifluomethyl,pentafluoroethyl, alkyl of 1 to about 12 carbon atoms, aryl of about 6to about 14 carbon atoms, and aralkyl of about 6 to about 15 carbonatoms;

(b) A₂ is selected from the group consisting of ##STR3## wherein (i) mis 1, 2 or 3;

(ii) n is 0, 1, 2, 3 or 4;

(iii) R₂ is selected from the group consisting of alkyl of 1 to about 12carbon atoms, alkenyl of about 3 to about 6 carbon atoms, aryl of about6 to about 14 carbon atoms, aralkyl of about 6 to about 15 carbon atomsand aralkenyl of about 8 to about 15 carbons atoms; and

(iv) R₃ is selected from the group consisting of hydrogen, alkyl of 1 toabout 4 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkylof about 6 to about 15 carbon atoms, and alkyl of 1 to about 4 carbonatoms substituted with a substituent selected from the group consistingof --OH, --C(O)--OH, --C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃and --NH--S(O₂)--CH₃ ;

(c) A₃ is an amino acid residue of an amino acid selected from the groupconsisting of L-alanine, L-azetidinecarboxylic acid, glycine,L-isoleucine, L-leucine, L-lysine mono-substituted at its e-amino groupwith R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine, L-ornithinemono-substituted at its δ-amino group with R₂ --S(O₂)--, L-pipecolicacid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline wherein R₂ is as defined hereinabove; and

(d) A₄ is selected from the group consisting of ##STR4## wherein (i) pand q are each independently selected integers from 1 to 5, wherein thesum of p+q is 4 to 8;

(ii) R₄ is aryl of about 6 to about 14 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of alkyl of 1 to about 4 carbon atoms, alkoxy of 1to about 4 carbon atoms, --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH,--NO₂ and --CF₃ ;

(iii) R₅ is aryl of about 6 to 14 carbon atoms;

(iv) R₆ is selected from the group consisting of hydrogen and alkyl of 1to about 4 carbon atoms;

(v) R₇ is selected from the group consisting of

hydrogen;

alkyl of 1 to about 4 carbon atoms;

aryl of about 6 to about 14 carbon atoms which is optionally substitutedwith 1 or 2 substituents each independently selected from the groupconsisting of --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH, --NO₂,--CF₃, alkyl of 1 to about 4 carbon atoms, and alkoxy of 1 to about 4carbon atoms;

aralkyl of about 6 to about 15 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH,--NO₂, --CF₃, alkyl of 1 to about 4 carbon atoms, and alkoxy of 1 toabout 4 carbon atoms; and

alkyl of 1 to about 4 carbon atoms substituted with a substituentselected from the group consisting of --OH, --C(O)--OH, --C(O)--NH₂,--S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ; and

(vi) R₈ is selected from the group consisting of alkyl of 1 to about 12carbon atoms, aryl of about 6 to about 14 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,and aralkyl of about 6 to about 15 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,wherein X₃ and X₄ are independently selected from the group consistingof --C(O)--OH, ##STR5## (vii) r is 0, 1, 2 or 3; and pharmaceuticallyacceptable salts thereof.

In another aspect, the present invention is directed to a pharmaceuticalcomposition for treating coagulation disorders which comprises apharmaceutically acceptable carrier and a therapeutically effectiveamount of a compound of the present invention.

In yet another aspect, the present invention is directed to methods ofpreventing or treating in a mammal a condition characterized by abnormalthrombus formation.

One aspect of the present invention allows the stereoselective synthesiswhich yields the optically pure arginine ketoamides of formula I. Thus,in another aspect, the present invention is directed to intermediatesuseful for the preparation of the compounds of the present invention.These intermediates have the structure: ##STR6## wherein (a) B₁ isselected from the group consisting of R₉ --C(O)--, R₉ --O--C(O)--, R₉--NH--C(O)--, R₉ --S(O₂)--, R₉ --O--S(O₂)-- and R₉ --NH--S(O₂)--,wherein R₉ is selected from the group consisting of

alkyl of 1 to about 12 carbon atoms,

alkenyl of about 3 to about 6 carbon atoms,

aryl of about 6 to about 14 carbon atoms which is optionallymono-substituted with x₅ or optionally di-substituted with X₅ and X₆,

aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted with x₅ or optionally di-substituted with X₅ and X₆,

aralkenyl of about 8 to about 15 carbon atoms which is optionallymono-substituted with X₅ or optionally di-substituted with X₅ and X₆,

perfluoroalkyl of 1 to about 12 carbon atoms,

perfluoroaryl of about 6 to about 14 carbon atoms,

trimethylsilylalkyl of 4 to about 8 carbon atoms, ##STR7## wherein X₅and X₆ are independently selected from the group consisting of bromo,chloro, fluoro, Y₃ --, Y₃ --O--, Y₃ --O--C(O)--NH--, Y₃--O--C(O)--N(Y₄)--, (Y₃,Y₄)N--, Y₃ --C(O)--NH--, Y₃ --S--, Y₃ --S(O)--,Y₃ --S(O₂)--, Y₃ --O--S(O₂)--, NH₂ --S(O₂)-- and Y₃ --NH--S(O₂)--,wherein Y₃ and Y₄ are independently selected from the group consistingof trifluoromethyl, pentafluoroethyl, aryl of about 6 to about 14 carbonatoms, aralkyl of about 6 to about 15 carbon atoms and alkyl of 1 carbonatom to about 12 which is optionally mono-substituted with aralkyloxy ofabout 6 to about 15 carbon atoms;

(b) B₂ is selected from the group consisiting of ##STR8## wherein (i) sis 1, 2 or 3;

(ii) t is 0, 1, 2, 3 or 4;

(iii) R₁₀ is selected from the group consisting of alkenyl of about 3 toabout 6 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkylof about 6 to about 15 carbon atoms, aralkenyl of about 8 to about 15carbons atoms and alkyl of 1 to about 12 carbon atoms which isoptionally mono-substituted with aralkyloxy of about 6 to about 15carbon atoms; and

(iv) R₁₁ is selected from the group consisting of hydrogen, alkyl of 1to about 4 carbon atoms, aryl of about 6 to about 14 carbon atoms,aralkyl of about 6 to about 15 carbon atoms, and alkyl of 1 to about 4carbon atoms substituted with a substituent selected from the groupconsisting of --O--R₁₀, --C(O)--O--R₁₀, --C(O)--NH₂, --S--CH₃,--S(O)--CH₃, --S(O₂)--CH₃ and --NH--S(O₂)--CH₃ ;

(c) B₃ is an amino acid residue of an amino acid selected from the groupconsisting of L-alanine, L-azetidinecarboxylic acid, glycine,L-isoleucine, L-leucine, L-lysine mono-substituted at its e-amino groupwith R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine, L-ornithinemono-substituted at its δ-amino group with R₂ --S(O₂)--, L-pipecolicacid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline substituted at 4-hydroxy group with R₁₂--O--C(O)--, wherein R₁₂ is selected from the group consisting of alkylof 1 to about 12 carbon atoms and aralkyl of about 6 to about 15 carbonatoms; and

(d) B₄ is selected from the group consisting of ##STR9## wherein (i) uand v are each independently selected integers from 1 to 5, wherein thesum of u+v is 4 to 8;

(ii) R₁₃ is aryl of about 6 to about 14 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of alkyl of 1 to about 4 carbon atoms, alkoxy of 1to about 4 carbon atoms, --NH--C(O)--O--X₇, --C(O)--O--X₇, --C(O)--NH₂,fluoro, --O--X₇, --NO₂ and --CF₃ ;

(iii) R₁₄ is aryl of about 6 to about 14 carbon atoms;

(iv) R₁₅ is selected from the group consisting of hydrogen and alkyl of1 to about 4 carbon atoms;

(v) R₁₆ is selected from the group consisting of

hydrogen,

alkyl of 1 to about 4 carbon atoms,

aryl of about 6 to about 14 carbon atoms which is optionally substitutedwith 1 or 2 substituents each independently selected from the groupconsisting of --NH--C(O)--O--X₈, --C(O)--O--X₈, --C(O)--NH₂, fluoro,--O--X₈, --NO₂, --CF₃, alkyl of 1 to about 4 carbon atoms, and alkoxy of1 to about 4 carbon atoms,

aralkyl of about 6 to about 15 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of --NH--C(O)--O--X₉, --C(O)--O--X₉, --C(O)--NH₂,fluoro, --O--X₉, --NO₂, --CF₃, alkyl of 1 to about 4 carbon atoms, andalkoxy of 1 to about 4 carbon atoms, and

alkyl of 1 to about 4 carbon atoms substituted with a substituentselected from the group consisting of --O--X₁₀, --C(O)--O--X₁₀,--C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ;

(vii) R₁₇ is selected from the group consisting of alkyl of 1 to about 4carbon atoms and aralkyl of about 6 to 15 carbon atoms;

(viii) R₁₈ is alkyl of 1 to about 12 carbon atoms which is optionallymono-substituted with aralkyloxy of about 6 to about 15 carbon atoms;

(ix) R₁₉ is selected from the group consisting of hydrogen, aryl ofabout 6 to about 14 carbon atoms which is optionally mono-substitutedwith X₁₁ or optionally di-substituted with X₁₁ and X₁₂ ; and aralkyl ofabout 6 to about 15 carbon atoms which is optionally mono-substitutedwith X₁₁ or optionally di-substituted with X₁₁ and X₁₂ ; and

(x) w is 0, 1, 2, 3, 4 or 5;

wherein X₇, X₈, X₉ and X₁₀ are independently selected from the groupconsisting of alkyl of 1 to about 4 carbon atoms, aryl of about 6 toabout 14 carbon atoms and aralkyl of about 6 to about 15 carbon atoms;and wherein X₁₁ and X₁₂ are independently selected from the groupconsisting of --(C(O)--O--R₁₇, --S(O2)--O--R₁₇, ##STR10## Definitions

In accordance with the present invention and as used herein, thefollowing terms are defined with the following meanings, unlessexplicitly stated otherwise.

The term "amino acid" refers to both natural and unnatural amino acidsin either their L- or D- forms. Natural amino acids include alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine andvaline. For example, unnatural amino acids include, but are not limitedto azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid,β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyricacid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyricacid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminoisobutyricacid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine,3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine,N-methylglycine, N-methylisoleucine, N-methylvaline, norvaline,norleucine, ornithine and pipecolic acid.

The term "amino acid residue" refers to --NH--CH(R)--CO--, wherein R isthe side chain group distinguishing each amino acid. For cyclic aminoacids, the residue is ##STR11## wherein x is 1, 2 or 3 representing theazetidinecarboxylic acid, proline or pipecolic acid residues,respectively.

The term "alkyl" refers to saturated aliphatic groups includingstraight-chain, branched-chain and cyclic groups.

The term "alkenyl" refers to unsaturated hydrocarbyl group which containat least one carbon-carbon double bond and includes straight-chain,branched-chain and cyclic groups.

The term "aryl" refers to aromatic groups which have at least one ringhaving a conjugated pi electron system and includes carbocyclic aryl,heterocyclic aryl and biaryl groups, all of which may be optionallysubstituted.

The term "aralkyl" refers to an alkyl group substituted with an arylgroup. Suitable aralkyl groups include benzyl, picolyl, and the like,all of which may be optionally substituted.

The term "aralkenyl refers to an alkenyl group substituted with an arylgroup. Suitable aralkenyl groups include styrenyl and the like, all ofwhich may be optionally substituted.

The term "alkoxy" refers to the group --OR wherein R is alkyl.

The term "alkenyloxy" refers to the group --O--R wherein R is alkenyl.

The term "aryloxy" refers to the group --O--R wherein R is aryl.

The term "aralkyloxy" refers to the group --O--R wherein R is aralkyl.

The term "alkylene" refers to a divalent straight chain or branchedchain saturated aliphatic radical.

The term "alkylenecarboxy" refers to the group -alk-COOH where alk isalklene.

The term "carboxamide" refers to the group --C(O)--NH₂.

The term "alkylenecarboxamide" refers to the group -alk-C(O)NH₂ wherealk is alkylene.

The term "alkylenehydroxy" refers to the group -alk-OH wherein alk isalkylene.

The term "methylene" refers to --CH₂ --.

The term "perfluoroalkyl refers to an alkyl group wherein each hydrogenis replaced by a fluoro. Suitable perfluoroalkyl groups includeperfluoromethyl (having the structure of CF₃ --) and perfluroethyl(having the structure of CF₃ --CF₂ --) and the like.

The term "perfluoroakyl refers to an aryl group wherein each hydrogen isreplaced by a fluoro. Suitable perfluoroaryl groups includeperfluorophenyl (having the formula of ##STR12## and 2-perfluoronaphthyl(having the formula of ##STR13## and the like.

In addition, the following abbreviations stand for the following:

"Bn" refers to benzyl.

"Boc" refers to t-butoxycarbonyl.

"Boc₂ O refers di-t-butyldicarbonate.

"BoCAsp^(Bn) -OH" refers to N-Boc-L-aspartic acid- (β-benzyl ester).

"BocPro-OH" refers to N-Boc-L-proline.

"Bom" refers to benzyloxymethyl.

"BOP" refers tobenzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate.

"Brine" refers to an aqueous saturated solution of sodium chloride.

"CH₂ Cl₂ " refers to dichloromethane.

"CH₃ CN" refers to acetonitrile.

"DCA" refers to dichloroacetic acid.

"DMF" refers to dimethylformamide.

"DMSO" refers to dimethylsulfoxide.

"EDC" refers to ethyl-3-(3-dimethylamino)propylcarbodiimidehydrochloride salt.

"HOBt" refers to 1-hydroxybenzotriazole.

"HCl" refers to hydrochloric acid.

"HF" refers to hydrofluoric acid.

"HPLC" refers to high pressure liquid chromatography.

"KOH" refers to potassium hydroxide.

"MeOH" refers to methanol.

"NaCO₃ " refers to sodium carbonate.

"NEt₃ " refers to triethylamine.

"NMM" refers to 4-methylmorpholine.

"Pd/C" refers to palladium on carbon.

"PhMe" refers to toulene.

"POCl₃ " refers to phosphorous oxychloride

"TFA" refers to trifluoroacetic acid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, i represents potassium cyanide, potassium bicarbonate, water; iirepresents HCl/water/dioxane; iii represents dry HCl/methanol; ivrepresents Boc₂ O/THF/NaHCO₃ /H₂ O/; v represents lithiumhydroxide/methanol/water; vi represents Dowex-50 acid form; viirepresents B₄ -NH₂ /BOP/DMF where B₄ is as defined in connection withformula I; viii represents TFA/methylene chloride; ix representsBoc-Pro-OH/BOP/DMF; x represents Boc-Asp^(Bn) -OH/BOP/DMF; xi representsmodified Moffatt conditions; and xii represents either H₂ /Pd on carbonor HF/anisole.

DETAILED DESCRIPTION OF THE INVENTION

Preferred Compounds

The compounds of the present invention can be divided conceptually intoparts as shown in the following formula Ia: ##STR14## P₁ corresponds toan arginine residue. P₂ corresponds to an amino acid residue such as aproline residue, trans-4-hydroxyproline residue, glycine residue,isoleucine residue and other of the above-specified amino acid residues.P₃ corresponds to an amino acid residue such as an aspartic acidresidue, aspartic acid ester residue, glycine amino acid residue,glutamic acid residue, glutamic acid ester residue, methionine sulfoneresidue, alanine residue which is β-substituted with a substituted orunsubstituted tetrazole, and other of the above-specified amino acids orderivatives thereof. In a particular compound, the A₁ and A₄ groups forP₄ and P₁ ' respectively are selected depending on the specific enzymeto be selectively inhibited.

Among other factors, in one aspect, the present invention is based onour surprising finding that the compounds of formula I which include atthe P₃ position an aspartic acid residue, suitable ester derivative ofaspartic acid, methionine sulfone residue or alanine residueβ-substituted with a substituted or unsubstituted tetrazole are highlyactive inhibitors of in vitro coagulation. Certain of these compoundsexhibit IC₅₀ 's in an assay of thrombin inhibition of less than 10 nm.(See Example A).

The compounds of formula I possessing a negative charge at physiologicalpH are thought to have specific advantages as in vivo antithromboticagents. The negative charge may be incorporated into these molecules inseveral ways. Compounds of formula I which include at the P₃ positionthe aspartic acid residue or an alanine residue β-substituted with anunsubstituted tetrazole directly possess such a such a negative charge.Compounds of formula I having at the P3 position, a methionine sulfoneresidue, suitable ester derivatives of aspartic acid or suitablederivatives of the alanine residue β-substituted with a substitutedtetrazole derivatives, may be advantageously derivatized elsewhere withan ionizable group which at physiological pH would yield a negativecharge. Such negatively charged groups would include the carboxy group,sulfonate group or unsubstituted tetrazole which in one approach may beconveniently introduced at the P₁ ' position.

Suitable ester derivatives of aspartic acid include those that can becleaved in vivo to yield the corresponding aspartic acid derivative.Such esters are believed to exhibit improved bioavailability and to havea longer half life in the circulation. The preferred compounds offormula I have A₁ groups which preferably comprise hydrophobic groupswhich have been selected so as to enhance potency and/or selectivity ofthese compounds.

Compounds of the present invention include those represented by formulaI. ##STR15##

The compounds of the present invention include those wherein A₁ is R₁--C(O)--, R₁ --O--C(O)--, R₁ --NH--C(O)--, R₁ --S(O₂)--, R₁ --O--S(O₂)--or R₁ --NH--S(O₂)--. Preferred compounds include those wherein A₁ is R₁--C(O)--, R₁ --O--C(O)-- or R₁ --S(O₂)--.

The compounds of the present invention include those wherein R₁ is analkyl of 1 to about 12 carbon atoms; alkenyl of about 3 to about 6carbon atoms; aryl of about 6 to about 14 carbon atoms which isoptionally mono-substituted with X₁ or optionally di-substituted with X₁and X₂ ; aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂ ;aralkenyl of about 8 to about 15 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂ ;perfluoroalkyl of 1 to about 12 carbon atoms; perfluoroaryl of about 6to about 14 carbon atoms; trimethylsilylalkyl of 4 to about 8 carbonatoms; ##STR16## wherein X₁ and X₂ are each independently selected fromthe group consisting of bromo, chloro, fluoro, Y₁ --, HO--, Y₁ --O--,NH₂ --, Y₁ --NH--, (Y₁,Y₂)N--, Y₁ --C(O)--NH--, HS--, Y₁ --S--, Y₁--S(O)--, Y₁ --S(O₂)--, HO--S(O₂)--, Y₁ --O--S(O₂)--, NH₂ --S(O₂)-- andY₁ --NH--S(O₂)--, wherein Y₁ and Y₂ are independently selected from thegroup consisting of trifluomethyl, pentafluoroethyl, alkyl of 1 to about12 carbon atoms, aryl of about 6 to about 14 carbon atoms, and aralkylof about 6 to about 15 carbon atoms.

Preferred compounds include those wherein R₁ is alkyl of 1 to about 12carbon atoms; aryl of about 6 to about 14 carbon atoms which isoptionally mono-substituted with X₁ or optionally di-substituted with X₁and X₂ ; or aralkyl of about 6 to about 15 carbon atoms which isoptionally mono-substituted with X₁ or optionally di-substituted with X₁and X₂. Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl,propyl, 2-methylpropyl, 2,2-dimethylpropyl, butyl, 3 -methylbutyl,1-propylbutyl, pentyl, hexyl, cyclopentyl, cyclopentylmethyl,cyclohexyl, cyclohexylmethyl, adamantyl and adamantylmethyl. Suitablearyl groups include phenyl, naphthyl, biphenyl, 2-thienyl, 2-pyrrolyland 2-furyl. Suitable aralkyl groups include phenylmethyl,diphenylmethyl, biphenyl, biphenylmethyl, naphthyl, naphthylmethyl,α-phenylmethylphenyl and 2-phenylethylene.

Especially preferred compounds include those wherein R₁ is1,1-dimethylethyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl,1-propylbutyl, phenylmethyl or naphthyl.

The compounds of the present invention include those wherein A₂ is##STR17## wherein m is 1, 2 or 3; n is 0, 1, 2, 3 or 4; R₂ is alkyl of 1to about 12 carbon atoms, carbon atoms, aryl of about 6 to about 14carbon atoms, aralkyl of about 6 to about 15 carbon atoms, or aralkenylof about 8 to about 15 carbons atoms; and R₃ is hydrogen, alkyl of 1 toabout 4 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkylof about 6 to about 15 carbon atoms, or alkyl of 1 to about 4 carbonatoms substituted with a substituted selected from the group consistingof --OH, --C(O)--OH, --C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃and --NH--S(O₂)--CH₃.

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable alkenyl groupsinclude 2-propenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 5-hexenyl and2-cyclopentenyl. Suitable aryl groups include phenyl, naphthyl,biphenyl, pyridyl, 2-thienyl, 2-pyrrolyl and 2-furyl. Suitable aralkylgroups include phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl,naphthyl, naphthylmethyl, α-phenylmethylphenyl and 2-phenylethylene.

Preferred compounds include those wherein A₂ is --(CH₂)_(m)--C(O)--O--H, ##STR18## --(CH₂)_(m) --C(O)--O--R₂ ##STR19## --(CH₂)_(m)--S(O₂)--R₂, or hydrogen; wherein m is 1 or 2 and if there is an R₂, itis alkyl of 1 to about 12 carbon atoms.

Especially preferred compounds include those wherein m is 1 and if thereis an R₂, it is methyl.

The compounds of the present invention include those wherein A₃ is anamino acid residue of L-alanine, L-azetidinecarboxylic acid, glycine,L-isoleucine, L-leucine, L-lysine mono-substituted at its e-amino groupwith R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine, L-ornithinemono-substituted at its δ-amino group with R₂ --S(O₂)--, L-pipecolicacid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline, wherein R₂ is alkyl of 1 to about 12 carbonatoms, alkenyl of about 3 to about 6 carbon atoms, aryl of about 6 toabout 14 carbon atoms, aralkyl of about 6 to about 15 carbon atoms andaralkenyl of about 8 to about 15 carbons atoms.

Preferred compounds include those wherein A₃ is glycine, L-isoleucine orproline.

Especially preferred compounds include those wherein A₃ is proline.

The compounds of the present invention include those wherein A₄ is##STR20## wherein (i) p and q are each independently selected integersfrom 1 to 5, wherein the sum of p+q is 4 to 8;

(ii) R₄ is aryl of about 6 to about 14 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of alkyl of 1 to about 4 carbon atoms, alkoxy of 1to about 4 carbon atoms, --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH,--NO₂ and --CF₃ ;

(iii) R₅ is aryl of about 6 to 14 carbon atoms;

(iv) R₆ is selected from the group consisting of hydrogen and alkyl of 1to about 4 carbon atoms;

(v) R₇ is selected from the group consisting of hydrogen; alkyl of 1 toabout 4 carbon atoms; aryl of about to about 14 carbon atoms which isoptionally substituted with 1 or 2 substituents each independentlyselected from the group consisting of --NH₂, --C(O)--OH, --C(O)--NH₂,fluoro, --OH, --NO₂, --CF₃, alkyl of 1 to about 4 carbon atoms, andalkoxy of 1 to about 4 carbon atoms; aralkyl of about 6 to about 15carbon atoms which is optionally substituted with 1 or 2 substituentseach independently selected from the group consisting of --NH₂,--C(O)--OH, --C(O)--NH₂, fluoro, --OH, --NO₂, --CF₃, alkyl of 1 to about4 carbon atoms, and alkoxy of 1 to about 4 carbon atoms; and alkyl of 1to about 4 carbon atoms substituted with a substituent selected from thegroup consisting of --OH, --C(O)--OH, --C(O)--NH₂, --S--CH₃,--S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ; and

(vi) R₈ is selected from the group consisting of alkyl of 1 to about 12carbon atoms, aryl of about 6 to about 14 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,and aralkyl of about 6 to about 15 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,wherein X₃ and X₄ are independently selected from the group consistingof --C(O)--O--, --S(O₂)--OH, ##STR21## and (vii) r is 0, 1, 2 or 3

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropel, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable alkenyl groupsinclude 2-propenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 5-hexenyl and2-cyclopentenyl. Suitable aryl groups include phenyl, naphthyl,biphenyl, pyridyl, 2-thienyl, 2-pyrrolyl and 2-furyl. Suitable aralkylgroups include phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl,naphthyl, naphthylmethyl, α-phenylmethylphenyl and 2-phenylethylene.Suitable substituted alkyls which include carboxymethyl, carboxyethyl,carboxypropyl, carboxybutyl, carboxypentyl and carboxyhexyl.

Preferred compounds include those wherein A₄ is ##STR22##

Especially preferred compounds include those wherein R₆, R₇ or both arehydrogen, r is 0, and where applicable R₈ is benzyl or 2-phenylethyl.

Preferred compounds of the present invention include ##STR23##

According to another aspect, the present invention is directed topharmaceutically acceptable salts of the compounds of formula I."Pharmaceutically acceptable salt" includes within its definition saltsof the compounds of the present invention derived from the combinationof a such compounds and an organic or inorganic acid. In practice, theuse of the salt form amounts to use of the base form. The compounds ofthe present invention are useful in both free base and salt form, withboth forms being considered as being within the scope of the presentinvention. These salts include acid addition salts, for example, saltsof hydrochloric acid, hydrobromic acid, acetic acid, benzene sulfonicacid and other suitable acid addition salts.

In yet another aspect, the present invention is directed to compoundsuseful as intermediates for the preparation of compounds represented byformula I. These intermediates are represented by formula II: ##STR24##The compounds of the present invention include those wherein B₁ is R₉--C(O)--, R₉ --O--C(O)--, R₉ --H--C(O)--, R₉ --S(O₂)--, R₉ --O--S(O₂)--or R₉ --NH--S(O₂)--. Preferred compounds include those wherein B₁ is R₉--C(O)--, R₉ --O--C(O)-- or R₉ --S(O₂)--.

The compounds of the present invention include those wherein is alkyl of1 to about 12 carbon atoms; alkenyl of about 3 to about 6 carbon atoms;aryl of about 6 to about 14 carbon atoms which is optionallymono-substituted with X₅ or optionally di-substituted with X₅ and X₆ ;aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted with X₅ or optionally di-substituted with X₅ and X₆ ;aralkenyl of about 8 to about 15 carbon atoms which is optionallymono-substituted with X₅ or optionally di-substituted with X₅ and X₆ ;perfluoroalkyl of 1 to about 12 carbon atoms; perfluoroaryl of about 6to about 14 carbon atoms; trimethylsilylalkyl of 4 to about 8 carbonatoms, ##STR25## wherein X₅ and X₆ are each independently selectedindependently and are bromo, chloro, fluoro, Y₃ --, Y₃ --O--, Y₃--O--C(O)--NH--, Y₃ --O--C(O)--N(Y₄)--, (Y₃,Y₄)N--, Y₃ --C(O)--NH--, Y₃--S--, Y₃ --S(O)--, Y₃ --S(O₂)--, Y₃ --O--S(O₂)--, NH₂ --S(O₂)-- or Y₃--NH--S(O₂)--, wherein Y₃ and Y₄ are independently selected and aretrifluoromethyl, pentafluoroethyl, aryl of about 6 to about 14 carbonatoms, aralkyl of about 6 to about 15 carbon atoms and alkyl of 1 carbonatom to about 12 which is optionally mono-substituted with aralkyloxy ofabout 6 to about 15 carbon atoms.

Preferred compounds include those wherein R₉ is alkyl of 1 to about 12carbon atoms; aryl of about 6 to about 14 carbon atoms which isoptionally mono-substituted with X₅ or optionally di-substituted with X₅and X₆ ; or aralkyl of about 6 to about 15 carbon atoms which isoptionally mono-substituted with X₅ or optionally di-substituted with X₅and X₆.

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable aryl groupsinclude phenyl, naphthyl, biphenyl, pyridyl, 2-thienyl, 2-pyrrolyl and2-furyl. Suitable aralkyl groups include phenylmethyl, diphenylmethyl,biphenyl, biphenylmethyl, naphthyl, naphthylmethyl, α-phenylmethylphenyland 2-phenylethylene.

Especially preferred compounds include those wherein R₉ is1,1-dimethylethyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl,1-propylbutyl, phenylmethyl or naphthyl.

The compounds of the present invention include those wherein B₂ is##STR26## wherein s is 1, 2 or 3; t is 0, 1, 2, 3 or 4; R₁₀ is alkenylof about 3 to about 6 carbon atoms, aryl of about 6 to about 14 carbonatoms, aralkyl of about 6 to about 15 carbon atoms, aralkenyl of about 8to about 15 carbons atoms, or alkyl of 1 to about 12 carbon atoms whichis optionally mono-substituted with aralkyloxy of about 6 to about 15carbon atoms; and R₁₁ is hydrogen, alkyl of 1 to about 4 carbon atoms,aryl of about 6 to about 14 carbon atoms, aralkyl of about 6 to about 15carbon atoms, or alkyl of 1 to about 4 carbon atoms substituted with asubstituent selected from the group consisting of --O--R₁₀,--C(O)--O--R₁₀, --C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃ and--NH--S(O₂)--CH₃.

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable alkenyl groupsinclude 2-propenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 5-hexenyl and2-cyclopentenyl. Suitable aryl groups include phenyl, naphthyl,biphenyl, pyridyl, 2-thienyl, 2-pyrrolyl and 2-furyl. Suitable aralkylgroups include phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl,naphthyl, naphthylmethyl, α-phenylmethylphenyl and 2-phenylethylene.Suitable aralkyloxy groups include benzyloxymethyl.

Preferred compounds include those wherein B₂ is hydrogen, R₁₀ --,--(CH₂)_(s) --C(O)--O--R₁₀, ##STR27## or --(CH₂)_(s) --S(O₂)--R₁₀,wherein s is 1 or 2 and R₁₀ is alkyl of 1 to about 12 carbon atoms whichis optionally mono-substituted with aralkyloxy of about 6 to about 15carbon atoms.

Especially preferred compounds include those wherein s is 1 and R₁₀ ismethyl or benzyloxymethyl.

The compounds of the present invention include those wherein B₃ is anamino acid residue of L-alanine, L-azetidinecarboxylic acid, glycine,L-isoleucine, L-leucine, L-lysine mono-substituted at its ε-amino groupwith R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine, L-ornithinemono-substituted at its δ-amino group with R₂ --S(O₂)--, L-pipecolicacid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline substituted at 4-hydroxy group with R₁₂--O--C(O)--, wherein R₁₂ is alkyl of 1 to about 12 carbon atoms oraralkyl of about 6 to about 15 carbon atoms.

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable aralkyl groupsinclude phenylmethyl, diphenylmethyl, biphenyl, biphenylmethyl,naphthyl, naphthylmethyl, α-phenylmethylphenyl and 2-phenylethylene.

Preferred compounds include those wherein B₃ is glycine, L-isoleucine orproline.

Especially preferred compounds include those wherein B₃ is proline.

The compounds of the present invention include those wherein B₄ is##STR28## wherein u and v are each independently selected integers from1 to 5, where the sum of u+v is 4 to 8;

R₁₃ is aryl of about 6 to about 14 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of alkyl of to about 4 carbon atoms, alkoxy of 1 toabout 4 carbon atoms, --NH--C(O)--O--X₇, --C(O)--O--X₇, --C(O)--NH₂,fluoro, --O--X₇, --NO₂ and --CF₃ ;

R₁₄ is aryl of about 6 to about 14 carbon atoms;

R₁₅ is hydrogen or alkyl of 1 to about 4 carbon atoms;

R₁₆ is

hydrogen;

alkyl of 1 to about 4 carbon atoms;

aryl of about 6 to about 14 carbon atoms which is optionally substitutedwith 1 or 2 substituents each independently selected from the groupconsisting of --NH--C(O)--O--X₈, --C(O)--O--X₈, --C(O)--NH₂, fluoro,--O--X₈, --NO₂, --CF₃ ; alkyl of 1 to about 4 carbon atoms and alkoxy of1 to about 4 carbon atoms;

aralkyl of about 6 to about 15 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of --NH--C(O)--O--X₉, --C(O)--O--X₉, --C(O)--NH₂,fluoro, --O--X₉, --NO₂, --CF₃, alkyl of 1 to about 4 carbon atoms, andalkoxy of 1 to about 4 carbon atoms; and

alkyl of 1 to about 4 carbon atoms substituted with a substituentselected from the group consisting of --O--X₁₀, --C(O)--O--X₁₀,--C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ;wherein X₇, X₈, X₉ and X₁₀ are independently selected from the groupconsisting of alkyl of 1 to about 4 carbon atoms, aryl of about 6 toabout 14 carbon atoms and aralkyl of about 6 to about 15 carbon atoms;

R₁₇ is alkyl of 1 to about 4 carbon atoms or aralkyl of about 6 to 15carbon atoms;

R₁₈ is alkyl of 1 to about 12 carbon atoms which is optionallymono-substituted with aralkyloxy of about 6 to about 15 carbon atoms;

R₁₉ is hydrogen; aryl of about 6 to about 14 carbon atoms which isoptionally mono-substituted with X₁₁ or optionally di-substituted withX₁₁ and X₁₂ ; or aralkyl of about 6 to about 15 carbon atoms which isoptionally mono-substituted with X₁₁ or optionally di-substituted withX₁₁ and X₁₂ ; wherein X₁₁ and X₁₂ are independently selected from##STR29## and w is 0, 1, 2, 3, 4 or 5.

Suitable alkyl groups include methyl, ethyl, 1,1-dimethylethyl, propyl,2-methylpropyl, 2,2-dimethylpropyl, butyl, 3-methylbutyl, 1-propylbutyl,pentyl, hexyl, cyclopentyl, cyclopentylmethyl, cyclohexyl,cyclohexylmethyl, adamantyl and adamantylmethyl. Suitable aryl groupsinclude phenyl, naphthyl, biphenyl, pyridyl, 2-thienyl, 2-pyrrolyl and2-furyl. Suitable aralkyl groups include phenylmethyl, diphenylmethyl,biphenyl, biphenylmethyl, naphthyl, naphthylmethyl, α-phenylmethylphenyland 2-phenylethylene. Suitable alkoxy groups include methoxy, ethyloxy,propyloxy, butyloxy, isobutyloxy and pentyloxy, hexyloxy. Suitablearalkyloxy groups include benzyloxymethyl.

Preferred compounds include those wherein B₄ is ##STR30##

Especially preferred compounds include those wherein R₁₅, R₁₆ or bothare hydrogen, w is 0, and where applicable R₁₇ is benzyl, R18 isbenzyloxymethy or methyl, and R19 is benzyl or 2-phenylethyl.

Preparation of Preferred Compounds

The preferred compounds of formula I may be conveniently prepared byliquid phase methods.

One method of synthesizing the compounds of formula I comprisesconverting the α-amino protected amino acid to an "activated" derivativewherein its carboxyl group is rendered more susceptible to reaction withthe free N-terminal α-amino group of the target amino acid or peptidehaving an associated α-keto amide functionality. For example, the freecarboxyl of the α-amino protected (N-protected) amino acid can beconverted to a mixed anhydride by reaction of a N-protected amino acidwith ethyl choloroformate, pivaloyl chloride or like acid chlorides.Alternatively, the carboxyl of the α-amino protected amino acid can beconverted to an active ester such as a 2,4,5-trichloropheyl ester, apentachlorophenol ester, a pentafluorophenyl ester, a p-nitrophenylester, a N-hydroxysuccinimide ester, or an ester formed from1-hydroxybenzotriazole.

Another coupling method involves use of a suitable coupling agent suchas N,N'-dicyclohexylcarbodiimide or N,N'-diisopropyl-carbodiimide. Otherappropriate coupling agents are disclosed in E. Gross & J. Meinenhofer,The Peptides: Analysis, Structure, Biology, Vol. I: Major Methods ofPeptide Bond Formation (Academic Press, New York, 1979).

The α-amino group of the target amino acid or peptide having anassociated α-keto amide functionality employed in the synthesis of thecompounds of the present invention is selectively de-protected duringthe coupling reaction to prevent side reactions involving the reactive,unprotected, side chain functionalities. In addition, reactiveside-chain functional groups (e.g., amino, carboxyl, guanidinyl,hydroxyl, and sulfhydryl) must also be protected with suitableprotecting groups to prevent chemical reaction of those groups fromoccurring during both the initial and subsequent coupling steps.Suitable protecting groups, known in the art, are described in E. Gross& J. Meienhofer, The Peptides: Analysis, Structure, Biology, Vol. 3:Protection of Functional Groups in Peptide Synthesis (Academic Press,New York, 1981).

In selecting suitable α-amino and reactive side-chain protecting groupsto be used during synthesis of the compounds of formula I, the followingconsiderations may be determinative. An α-amino protecting group should:(a) render the α-amino function inert (i.e., non-reactive) under theconditions employed in the coupling reaction, (b) be readily removableafter the coupling reaction under conditions that will not removeside-chain protecting groups and will not alter the structure of thepeptide fragment, and (c) minimize or eliminate the possibility ofracemization upon activation immediately prior to coupling. An aminoacid side-chain protecting group should: (a) render the protected sidechain functional group inert under the conditions employed in thecoupling reaction, (b) be stable under the conditions employed inremoving the α-amino protecting group, and (c) be readily removable uponcompletion of the desired peptide under reaction conditions that willnot alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for liquid phase peptide synthesis may vary inreactivity with the agents employed for their removal. For example,certain protecting groups such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl, t-amyloxycarbonyl, adamantyl-oxycarbonyl, andp-methoxybenxyloxycarbonyl are less labile and require moderately strongacids, such as trifluoroacetic, hydrochloric, or boron trifluoride inacetic acid, for their removal. Still other protecting groups, such asbenxyloxycarbonyl, halobenxyloxycarbonyl, p-nitrobenzyloxycarbonylcycloalkyloxycarbonyl, and isopropyloxycarbonyl, are even less labileand require stronger acids, such as hydrogen fluoride, hydrogen bromide,or boron trifluoroacetate in trifluoroacetic acid, for their removal.

Examples of amino acid protecting groups which are conventionally usedinclude the following:

(1) For an α-amino group: (a) aromatic urethane-type protecting groups,such as fluorenylmethyloxycarbonyl (FMOC); (b) aliphatic urethane-typeprotecting groups, such as t-butyloxycarbonyl, t-amyloxycarbonyl,isopropyloxycarbonyl, 2-(p-biphenylyl)isopropyloxycarbonyl,allyloxycarbonyl and the like; (c) cycloalkyl urethane-type protectinggroups, such as cyclo-pentyloxycarbonyl, adamantyloxycarbonyl, andcyclohexyloxy-carbonyl; and (d) allyloxycarbonyl. Preferred α-aminoprotecting groups include t-butyloxycarbonyl orfluorenylmethyloxycarbonyl.

(2) For the side chain amino group present in lysine: protecting groupsinclude any of the groups mentioned above in (1) such ast-butyloxycarbonyl, p-chlorobenzyloxycarbonyl, etc.

(3) For the guanidino group of arginine: protecting groups preferablyinclude nitro, carbobenzyloxy, or2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,3,6-trimethyl-4-methoxyphenylsulfonyl.

(4) For the hydroxyl group of serine, threonine, or tyrosine: protectinggroups include, for example, t-butyl; benzyl; substituted benzyl groups,such as p-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl,and 2,6-dichlorobenzyl.

(5) For the carboxyl group of aspartic acid or glutamic acid: protectinggroups include, for example, by esterification using groups such ast-butyl, indan-5-yl or preferably benzyl.

(6) For the imidazole nitrogen of hystidine: suitable protecting groupsinclude the benzyloxymethyl group

(7) For the phenolic hydroxyl group of tyrosine: protecting groups suchas tetrahydropyranyl, tert-butyl, trityl, benzyl, chlorobenzyl,4-bromobenzyl, and 2,6-dichlorobenzly are suitably employed. Thepreferred protecting group is bromo-benzyloxycarbonyl.

(8) For the side chain sulfhydryl group of cysteine: trityl ispreferably employed as a protecting group.

Starting materials used in the preparation of these compounds arereadily available from commercial sources as Aldrich, Bachem BioScienceInc., Nova Biochemicals, and Sigma.

According to one suitable reaction scheme, the compounds of formula Iare prepared according to the following protocol. The α-amino protectinggroup is removed from a t-butyloxycarbonyl-protected amino acid orpeptide having an associated α-keto amide functionality, such as byusing trifluoroacetic acid in methylene chloride or trifluoroacetic acidalone. The deprotection is carried out at a temperature of from about 0°C. to about ambient temperature. Other suitable cleaving reagents, forremoval of specific α-amino protecting groups, such as HCl in dioxane,may be used.

After the α-amino protecting group is removed from the amino acid orpeptide, the desired α-amino and side-chain protected amino acid iscoupled to the α-amino deprotected amino acid or peptide. Additionalα-amino and side chain protected amino acids are coupled in a stepwisemanner in the desired order until the desired sequence has beencompleted. As an alternative to adding each amino acid separately duringthe synthesis, several amino acids may be coupled to one another to givea peptide fragment prior to their coupling to the target amino acidanalog. After the coupling steps are complete, the product peptideanalog is deprotected to give the compound of formula I. Selection of anappropriate coupling reagent is within the skill of the art.Particularly suitable coupling reagents includeN,N'-dicyclohexylcarbodiimide, diispropylcarbodiimide or BOP.

The compounds of the present invention, represented in formula I above,are synthesized by a preferred liquid phase method as depicted in FIG. 1and described below using the intermediates represented by formula IIabove.

Step A: The aldehyde functionality of the protected arginine aldehyde 1is chemically replaced with an α-hydroxyacetic acid group to give theprotected α-hydroxycarboxylic acid analogue of arginine, 6. Examples 2to 4 describe a series of reactions exemplifying how the aldehyde groupmay be converted to an α-hydroxyacetic acid group.

Step B: The newly-introduced carboxy group on intermediate 6 is coupledusing BOP to any suitable substituted amine, exemplified by amines suchas 2-phenylethylamine in Example 5 or 3-phenylpropylamine as in Example18, or by a protected amino acid as in Example 25 to give the amide 7. Asuitable amine will be any sufficiently reactive amine in which otherreactive groups are protected.

Step C: Suitably protected amino acids or peptides or peptide analogsare coupled to 7 after its N-terminus is deprotected. Examples 6, 9, 14and 16 describe reactions coupling variousN-protected-aspartyl-(β-benzyl ester)prolyl derivatives to amide 7, togive derivatives containing the α-hydroxycarboxylic analogues ofarginine, as exemplified by Examples 6, 9, 12, 15, and 19.

Step D: The α-hydroxy group of the resulting derivatives is oxidized toa keto group under modified Moffatt conditions. Edwards et al., J. Am.Chem. Soc., 114: 1854 at 1861 (1992). This gives the correspondingα-ketoamide derivatives, which are examples of the intermediates of thepresent invention. Exemplars of these intermediates are the compounds ofExamples 7, 10, 13, 16 and 20.

Step E: The protecting groups on the α-ketoamide derivatives is removedby means of catalytic hydrogenation (H₂ /Pd on Carbon) or HFdeprotection using HF/anisole to give the compounds of the presentinvention. Exemplars of these compounds are the compounds of Examples 8,11, 14, 17 and 21.

Purification of the compounds of the present invention is typicallyachieved using conventional procedures such as preparative HPLC(including reversed phase HPLC) or other known chromatography, affinitychromatography (including monoclonal antibody columns) or countercurrentdistribution.

Utility and Formulation

The present invention provides the novel compounds of formula I, theirpharmaceutically acceptable salts and compositions prepared from them.These compounds and pharmaceutical compositions are useful as inhibitorsof coagulation proteases, both in vitro and in vivo. As discussed in theBackground and Introduction to the Invention, the formation of thrombincatalyzed by factor Xa is the penultimate reaction in the coagulationcascade and is a reaction common to both the intrinsic and extrinsiccoagulation pathways which terminate in the formation of a fibrin clot.Inhibitors of this and other activated coagulation factors wouldtherefore inhibit fibrin deposition, thrombus formation and theconsumption of coagulation proteins.

Inhibitors of activated coagulation proteases may be used aspharmacological agents for the treatment of thrombotic disordersincluding, myocardial infarction, unstable angina, disseminatedintravascular coagulation and associated complications resulting fromvenous thrombosis. These inhibitors may used as adjunctive orconjunctive agents to prevent recurrent thrombosis following enzymaticthrombolysis and percutaneous transluminal angioplasty. In addition,specific inhibitors of factor Xa may be useful in the supression ofmetastatic migration of certain tumor types as described by Tuszynski,G. P. et. al., "Isolation and characterization of antistasin, aninhibitor of metastasis and coagulation", J. Biol. Chem., 262: 9718-9723(1987) and Brankamp, R. G. et. al., "Ghilantens: anticoagulants,antimetastatic proteins from the South American leech Haementeriaghilianii"", J. Lab Clin. Med., 115: 89-97 (1990).

In mammals, in vivo uses would include administration of these compoundsand compositions as therapeutic agents to prevent the formation offibrin clots in blood vessels resulting from the presence of activatedcoagulation proteases, to prevent abnormal thrombus formation resultingfrom thrombotic disorders, and to prevent or treat the recurrentthrombus formation resulting from chemical or mechanical interventiondirected to clearing blocked vessels. Additionally, the compounds, theirsalts and various compositions derived therefrom may be useful astherapeutic agents for suppressing the metastatic migration of tumortypes in mammals.

The in vitro inhibitory activity of the compounds of the presentinvention may be demonstrated using an enzyme inhibition assay. The testcompound is dissolved in a suitable assay buffer to give a solutionhaving a concentration of test compound under assay conditions in therange of from 0 to about 100 mM. The enzyme whose activity is to beassayed is added to a solution containing a specified concentration ofthe test compound. After an incubation period, synthetic substrate forthe enzyme is added. The rate of substrate turnover is determinedspectrophotometrically at particular substrate concentrations. This datais used to determine an inhibition constant, Ki, for the test compound.Example A demonstrates that the compounds of Examples 8, 17, and 21 arepotent inhibitors of human α-thrombin, having Ki's of 11, 1.5 and 5.5nanomolar, respectively. These assay results demonstrate that thecompounds of formula I are active as inhibitors of thrombin in vitro.These assays are also considered to be indicative of in vivo activity.

The in vivo inhibitory activity of the compounds of formula I in a ratmodel of acute thrombosis was demonstrated. (See Example B). The testcompound was dissolved in a suitable diluent to give a test solution.The test solution is injected into a rat and the antithrombotic effectwas measured. Example B demonstrated that the compound of Example 8possessed antithrombic efficacy in vivo in a mammal. The compounds ofthe present invention are useful as inhibitors of thrombus formation.

Thus, in one aspect, the present invention is directed to methods forpreventing or treating a condition in mammals characterized by abnormalthrombus formation. The pharmaceutically effective amount of thecompound or composition of the present invention required as a dose willdepend on the route of administration, the type of mammal being treated,and the physical characteristics of the specific mammal underconsideration. The dose can be tailored to achieve optimal efficacy butwill depend on such factors as weight, diet, concurrent medication andother factors which those skilled in the medical arts will recognize.

In practicing the methods of the invention, the compounds orcompositions of the present invention can be used alone or incombination with one another, or in combination with other therapeuticor diagnostic agents. These compounds can be used in vivo, ordinarily ina mammal, preferably in a human, or in vitro. In employing them in vivo,the compounds or compositions can be administered to the mammal in avariety of ways, including parenterally, intravenously, subcutaneously,intramuscularly, colonically, rectally, nasally or intraperitoneally,employing a variety of dosage forms.

In another aspect the present invention is directed to pharmaceuticalcompositions prepared for storage and subsequent administration whichcomprise a therapeutically effective amount of a compound of formula Ior its pharmaceutically acceptable salt in a pharmaceutically acceptablecarrier or diluent.

The therapeutically effective amount of compound of formula I or itspharmaceutically acceptable salt which will be required as a dose willdepend on factors which include the route of administration, the type ofmammal being treated, and the physical characteristics of the specificmammal under consideration. One skilled in the medical art willappreciate that each composition of therapeutic drug has individualcharacteristics relating to drug absorption which may affect the amountto be compounded into a given pharmaceutically acceptable carrier ordiluent for a therapeutically effective dose. Ansel, H., "Dosage Formsand Routes of Administration", Introduction to Pharmaceutical DosageForms, 4th Edition, pp. 49-62, Lea & Febiger, Philadelphia (1985).

As will be readily apparent to one skilled in the art, the useful invivo dosage to be administered and the particular mode of administrationwill vary depending upon the age, weight and mammalian species treated,the particular compounds employed, and the specific use for which thesecompounds are employed. The determination of effective dosage levels,that is the dosage levels necessary to achieve the desired result, willbe within the ambit of one skilled in the art. Typically, applicationsof compound are commenced at lower dosage levels, with dosage levelbeing increased until the desired effect is achieved.

The dosage for the compounds of the present invention, theirpharmaceutically acceptable salts when compounded into pharmaceuticallyacceptable carriers or diluents can range broadly depending upon thedesired affects and the therapeutic indication. Typically, dosages willbe between about 0.01 mg/kg and 100 mg/kg body weight, preferablybetween about 0.01 mg/kg and 10 mg/kg body weight. Administration ispreferably parenteral, such as intravenous on a daily basis.

The compounds of formula I may be formulated and used as tablets,capsules or elixirs for oral administration; suppositories for rectaladministration; sterile solutions, suspensions for injectableadministration; and the like. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution or suspension in liquid prior to injection,or as emulsions. Suitable excipients are, for example, water, saline,dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate,cysteine hydrochloride, and the like. In addition, if desired, theinjectable pharmaceutical compositions may contain minor amounts ofnontoxic auxilliary substances, such as wetting agents, pH bufferingagents, and the like. Ansel, H., Introduction to Pharmaceutical DosageForms, 4th Edition, pp. 117-358, Lea & Febiger, Philadelphia (1985).Also, if desired, absorption enhancing preparations (e.g., liposomes)may be utilized.

Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).Preservatives, stabilizers, dyes and even flavoring agents may beprovided in the pharmaceutical composition. For example, sodiumbenzoate, sorbic acid and esters of p-hydroxybenzoic acid may be addedas preservatives. Id. at 1449. In addition, antioxidants and suspendingagents may be included. Id.

To assist in understanding the present invention, the following examplesare included which describe the results of a series of experiments. Thefollowing examples relating to this invention should not, of course, beconstrued as specifically limiting the invention. Variations of theinvention, now known or later developed, which would be within thepurview of one skilled in the art are considered to fall within thescope of the invention as described herein and hereinafter claimed.

Certain of the Examples, including Examples 1 to 29 illustrate thepreparation of the compounds of the present invention according to thesynthetic scheme depicted in FIG. 1. Example A illustrates the activityand use of the compounds of the present invention as an inhibitor ofthrombin. Example B illustrates the in vivo activity use of a compoundof the present invention in mammals as an antithrombotic agent.

EXAMPLES Example 1 Preparation of Alpha-N-t-butoxycarbonyl-N^(g)-nitroargininal ##STR31##

The following procedure for the synthesis ofalpha-t-butoxycarbonyl-N^(g) -nitro-argininal (Compound of Example 1) isa modification of the procedure of Fehrentz, J. A. and Castro, B.,Synthesis, 676 (1983).

BOC-N^(g) -nitroarginine was obtained from Calbiochem.N-methylpiperidine, N,O-dimethylhydroxylamine hydrochloride,isobutylchloroformate, and lithium aluminum hydride were obtained fromAldrich Chemical Company, Inc. pichloromethane, ethyl acetate, methanol,and tetrahydrofuran were obtained from Fisher Scientific Company.

11.4 mL of N-methylpiperidine was slowly added to a stirred suspensionof 9.17 g (94 mmole) of N,O-dimethylhydroxylamine hydrochloride in 75 mLof dichloromethane which had been cooled to about 0° C. The solution wasallowed to stir for 20 minutes and was kept cold for use in the nextstep.

In a separate flask, 30.0 g (94 mmole) of Boc-N^(g) -nitroarginine wasdissolved by heating in about 1400 mL of tetrahydrofuran and cooledunder nitrogen to 0° C. 11.4 mL of N-methylpiperidine and 12.14 mL (94mmole) of isobutylchloroformate were added and the mixture was stirredfor 10 minutes. The free hydroxylamine solution prepared above was addedin one portion and the reaction mixture was allowed stir overnight atroom temperature.

The resulting precipitate was removed by filtration and washed with 200mL of tetrahydrofuran. After concentrating the filtrates to about 150 mLunder vacuum, 200 mL of ethyl acetate was added, followed by ice to coolthe solution. The cooled solution was washed with two 75 mL portions of0.2N hydrochloric acid, two 75 mL portions of 0.5N sodium hydroxide, one75 mL portion of brine, then was dried with anhydrous magnesium sulfate.Upon concentration under vacuum, 22.7 g (70% yield) of solid BOC-N^(g)-nitroarginine N-methyl-O-methylcarboxamide was isolated. Thin layerchromatographic analysis in 9:1 dichloromethane/methanol (silica gel)showed one spot.

A flask was placed under a nitrogen atmosphere and cooled to -50° C.,then was charged with 70 mL (70 mmole) of 1M lithium aluminum hydride(in tetrahydrofuran) and 500 mL of dry tetrahydrofuran. A solutioncontaining 66 mmole of BOC-N^(g) -nitroarginineN-methyl-O-methylcarboxamide in 50 mL of dry tetrahydrofuran was slowlyadded while the temperature of the reaction mixture was maintained at-50° C. After allowing the reaction mixture to warm to 0° C. by removalof the cooling bath, it was cooled to -30° C., at which temperature, 100mL (0.2 mole) of 2N potassium bisulfate was added with stirring, over a10 to 15 minute period. The reaction mixture was then allowed to stir atroom temperature for 2 hours. After removal of the precipitate byfiltration, the filtrate was concentrated to 100 mL under vacuum. Theconcentrate was combined with 200 mL ethyl acetate, then washed with two50 mL portions of 1N hydrochloric acid, two ₅₀ mL portions of saturatedsodium bicarbonate, one 50 mL portion of brine, then was dried overanhydrous magnesium sulfate. The mixture was concentrated under vacuumto yield 13.6 g (70%) of the title compound.

Example 2 Preparation ofN-(Nitroguanidino-1-(S)-(cyanohydroxymethyl)butyl)-1-(1,1-dimethylethoxy)methanamide##STR32##

A solution of 25.2 g (83.1 mmoles) of alpha-Boc-N^(g) -nitro-argininal(the compound of Example 1) in 680 mL tetrahydrofuran was added to asolution of 136 g (1.36 moles) of potassium bicarbonate and 27.6 g (423mmoles) of potassium cyanide in 680 mL of water, This two phase mixturewas allowed to stir vigorously for thirty minutes, The stirring wasdiscontinued and the phases were separated, The aqueous phase wasextracted three times with 500 mL ethyl acetate, The tetrahydrofuranphase was diluted with 1000 mL of ethyl acetate, The organic phases werecombined and extracted successively with water and brine, This solutionwas dried over anhydrous magnesium sulfate and concentrated under vacuumto give 28.1 g of the above-identified product as a white foam, Thismaterial can be purified by flash chromatography (0 to 6% methanol indichloromethane) or carried through the next steps directly.

¹ H NMR (CD₃ OD) d 1.37 (s, 9H), 1.53 (m, 2H), 1.7 (m, 2H), 3.19 (m,2H), 3.65 (m, 1H), 4.29 (d, J=7 Hz, 0.35H), 4.48 (d, J=4 Hz, 0.65H).

Example 3 Preparation of6-Nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoicacid methyl ester ##STR33##

The 26.0 g (˜83 mmole) crude cyanohydrin (compound of the Example 2) wasdissolved in 450 mL dioxane, and 450 mL concentrated aqueoushydrochloric acid was slowly added with stirring. This addition wasaccompanied by vigorous gas evolution. This solution was heated toreflux and stirred for 15 hours. After this period of time, the reactionwas allowed to cool to room temperature and then concentrated undervacuum to a thick brown syrup of6-nitroguanidino-3-(S)-amino-2-hydroxyhexanoic acid hydrochloride salt(compound 3 of FIG. 1).

Crude amino acid 3 (of FIG. 1) from above was concentrated several timesfrom methanol under vacuum and then dissolved in 750 mL of saturatedanhydrous hydrochloric acid in methanol. This suspension was refluxedfor three hours, allowed to cool to room temperature and concentratedunder vacuum. This gave crude6-nitroguanidino-3-(S)-amino-2-hydroxyhexanoic acid methyl esterhydrochloride salt (compound 4 of FIG. 1) as a thick brown syrup. Thiswas used directly in the next step.

The amino ester (compound 4 of FIG. 1) from above was dissolved in amixture of 300 mL of saturated sodium bicarbonate and 300 mLtetrahydrofuran. This mixture was treated with di-t-butyldicarbonate (30g, 137 mmoles) and allowed to stir vigorously for 16 hours. Theresulting mixture was extracted with ethyl acetate (1000 mL). Theorganic layer was washed successively with water then brine, dried overanhydrous magnesium sulfate and concentrated to a small volume undervacuum. The product was purified by flash chromatography (0 to 10%methanol/dichloromethane) to give 13.5 g (49% yield) of theabove-identified product as an off-white foam. ¹ H NMR (CDCl₃) d 1.41and 1.45 (s, 9H), 1.7 (m, 4H), 3.2 (m, 2H), 3.82 and 3.84 (s, 3H), 4.10(m, 1H), 4.19 (bs, 0.65H), 4.33 (bs, 0.35H), 5.02 (d, J=10 Hz, 1H), 5.17(d, J=10 Hz, 1H).

Example 4 Preparation of6-Nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoicacid ##STR34##

A solution of the compound of Example 3 (5.0 g, 13.8 mmole) in 100 mL ofmethanol was treated with 17 mL of 1M lithium hydroxide. This solutionwas allowed to stir overnight and then treated with 20 mL of Dowex-50resin X8 400 (H⁺ form) in 50 mL of deionized water. This solution wasswirled for 15 minutes then passed through a 4×4 cm. column of the sameresin, the column was washed with 1:1 methanol:water and the combinedfiltrates were concentrated to dryness under vacuum. The residue wasdissolved in 100 mL acetonitrile and concentrated to dryness, thisprocess was repeated two more times to give 4.2 g (87 % yield) of theabove-identified compound as an off-white foam. ¹ H NMR (CD₃ OD) d 1.42and 1.42 (s, 9H), 1.7 (m, 4H), 3.3 (m, 2H), 3.95 (m, 1H), 4.19 (bs,0.65H), 4.33 (bs, 0.35H), 4.15 (d, J=1 Hz, 0.65H), 4.38 (d, J=4 Hz).

Example 5 Preparation of ##STR35##

A 1.05 g portion (2.90 mmole) of the compound of Example 3 was dissolvedwith stirring in 29 mL of methanol. To this solution was added 3.6 mL of1N aqueous sodium hydroxide. After 18 hours, thin layer chromatographicanalysis (10% methanol/dichloromethane) showed no more startingmaterial. The reaction was neutralized with 1.1 mL of 1N aqueoushydrochloric acid and concentrated under vacuum to dryness. Theresulting solid was then dissolved in 15 mL of dimethylformamide withstirring. This solution was treated successively with 0.364 mL (2.90mmole) 2-phenylethylamine, 0.86 mL (7.83 mmole) NMM and 1.41 g (3.19mmole) BOP. After 18 hours, thin layer chromatographic analysis (10%methanol/dichloromethane) showed no more material corresponding to theacid. The reaction mixture was poured into ethyl acetate (300 mL) andwashed successively with 1N aqueous hydrochloric acid (75 mL), water (75mL), saturated sodium bicarbonate (75 mL) and brine (75 mL). The organiclayer was dried over anhydrous magnesium sulfate and concentrated undervacuum to a foam. Flash chromatography (silica, 10%methanol/dichloromethane) afforded 1.18 g (90%) of a foam. R_(f) =0.33(two spots, 10% methanol/dichloromethane).

Example 6 Preparation of ##STR36##

To a solution of the compound of Example 5 (0.675 g, 1.49 mmole) in 17mL dichloromethane was added 17 mL of trifluoroacetic acid withstirring. After 30 minutes, thin layer chromatographic analysis (10%methanol/dichloromethane) showed no starting material. Thetrifluoroacetate salt was precipitated by adding 200 mL of diethyl etherand cooled in the freezer for 3 hours. The solid was removed byfiltration and rinsed with 75 mL diethyl ether. The resulting solid wasdissolved in 7 mL of dimethylformamide with stirring and this solutionwas treated with 0.627 g (1.49 mmole)α-N-(t-butoxycarbonyl)-L-aspartyl-(β-benzyl ester)-L-proline (thecompound of Example 24), 0.44 mL (4.02 mmole) NMM and 0.72 g (1.64mmole) BOP. After 18 hours, thin layer chromatographic analysis (10%methanol/dichloromethane) showed no more trifluoroacetate salt. Themixture was added to 300 mL of ethyl acetate and washed successivelywith 75 mL of 1N aqueous hydrochloric acid, 75 mL of water, 75 mL ofsaturated sodium bicarbonate and 75 mL of brine. The organic layer wasdried over anhydrous magnesium sulfate and concentrated under vacuum.Flash chromatography (silica, 3:1:9 hexane/methanol/dichloromethane)afforded 0.891 g (79%) of a foam. R_(f) =0.29 (10%methanol/dichloromethane).

Example 7 Preparation of ##STR37##

A 1.0 g portion (1.32 mmole) of the compound of Example 6 was dissolvedin 13 mL of dimethylsulfoxide with stirring. This solution was treatedwith 13 mL of toluene and 2.53 g (13.23 mmole) ofethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride followed by0.43 mL (5.29 mmole) of dichloroacetic acid. After 1 hour, thin layerchromatographic analysis (10% methanol/dichloromethane) showed a newspot and no starting material. The mixture was added to 500 mL of ethylacetate and washed with two 200 mL portions of water and 150 mL ofbrine. The organic layer was dried over anhydrous magnesium sulfate andconcentrated under vacuum. Flash chromatography (silica, 4:1:4hexane/methanol/dichloromethane) afforded 0.832 g (83%) of theabove-identified compound as a foam. R_(f) =0.32 (10%methanol/dichloromethane).

Example 8 Preparation of ##STR38##

A 0.512 g portion (0.667 mmole) of the compound of Example 7 wasdissolved in 50 mL of methanol. This solution was added to a Parr vesselcontaining 0.5 g 10% Pd/C, followed by 1.32 mL (1.32 mmole) 1N aqueoushydrochloric acid. The mixture was shaken under a 10 psig hydrogenatmosphere for 1.5 hours, after which HPLC (reverse phase, 1 mL/minute,40-80% acetonitrile/water with 0.1% trifluoroacetic acid, 20 minuteprogram, retention time=6.09 minute) showed complete reaction. Themixture was filtered, rinsed with 10 mL of methanol and concentratedunder vacuum. The resulting foam was purified by preparative HPLC(reverse phase, 50 mL/minute, 20-60% acetonitrile/water with 0.1%trifluoroacetic acid, 40 minute program). The appropriate fractions werecombined and the acetonitrile was removed under vacuum. The remainingliquid was frozen and lyophilized to afford 0.25 g (36%) of theabove-identified compound a white fluffy powder. Mass spectral analysisshowed the expected molecular ion at 617.3 (calc. 617.3).

Example 9 Preparation of ##STR39##

A solution of the compound of Example 6 (0.566 g, 0.749 mmole) in 14 mLof dichloromethane and 14 mL trifluoroacetic acid was allowed to stir atroom temperature. After 40 minutes, thin layer chromatographic analysis(10% methanol/dichloromethane) showed no starting material. Thetrifluoroacetate salt was precipitated by adding 200 mL diethyl ether.The mixture was allowed to cool in the freezer for 3 hours. The solidwas removed by filtration, rinsed with 75 mL of diethyl ether, dissolvedin 4 mL of dimethylformamide with stirring, and 0.062 mL (0.749 mmole)of 4-methylvaleric acid and 0.2 mL (2.02 mmole) of NMM were added,followed by 0.36 g of (0.82 mmole) of BOP. After 18 hours, thin layerchromatographic analysis (10% methanol/dichloromethane) showed no moretrifluoroacetate salt. The mixture was added to 300 mL of ethyl acetateand washed successively with 100 mL of 1N aqueous hydrochloric acid, 100mL of water, 100 mL of saturated sodium bicarbonate and 100 mL of brine.The organic layer was dried over anhydrous magnesium sulfate andconcentrated under vacuum to a foam. Flash chromatography (silica, 3:1:9hexane/methanol/dichloromethane) afforded 0.317 g (56%) of the abovecompound as a foam. R_(f) =0.32 (two spots, 10%methanol/dichloromethane).

Example 10 Preparation of ##STR40##

A 0.236 g portion of the compound of Example 9 (0.313 mmole) wasoxidized and worked up as described in Example 7. Flash chromatography(silica, 3:1:9 hexane/methanol/dichloromethane) of the concentratedorganic layer afforded 0.206 g (88%) of the above-identified compound asa foam. R_(f) =0.42 (10% methanol/dichloromethane).

Example 11 Preparation of ##STR41##

A 0.144 g portion of the compound of Example 10 (0.192 mmole) washydrogenated and worked up as described in Example 8. The concentratewas purified by preparative HPLC (reverse phase, 50 mL/minute, 20-80%acetonitrile/water with 0.1% trifluoroacetic acid, 40 minute program).The appropriate fractions were combined and the acetonitrile was removedunder vacuum. The remaining liquid was frozen and lyophilized to afford0.57 g (57%) the above-identified compound as a white fluffy powder.Mass spectral analysis showed the expected molecular ion at 615.3 (calc.615.3).

Example 12 Preparation of ##STR42##

The compound of Example 6 (0.68 g, 1.23 mmole) was converted to theabove-identified product using procedures as described in Example 9,using 3-methylcinnamic acid (in place of 4-methyl valeric acid). Flashchromatography (silica, 10% methanol/dichloromethane) afforded 0.582 g(59%) of the above-identified compound as a foam. R_(f) =0.34 (10%methanol/dichloromethane).

Example 13 Preparation of ##STR43##

A 0.541 g portion of the compound of Example 12 (0.541 g, 0.675 mmole)was oxidized and worked up as described in Example 7. Flashchromatography (silica, 4:1:4 hexane/methanol/dichloromethane) afforded0.462 g (85%) of the above-identified compound as a foam. R_(f) =0.37(10% methanol/dichloromethane).

Example 14 Preparation of ##STR44##

A 0.109 g portion of the compound of Example 13 (0.136 mmole) wastransferred to an hydrofluoric acid reaction vessel. Anisole (0.1 mL)and a stir bar were added. The vessel was flushed with nitrogen andhydrofluoric acid and cooled to -20° C. Hydrofluoric acid (3.0 mL) wasdistilled into the reaction vessel with stirring. After 30 minutes, thevessel was warmed to 0° C. and flushed with nitrogen. After 1 hour, thehydrofluoric acid was evaporated. The resulting material was extractedwith water then 20% acetic acid/water. Both aqueous layers were washedwith diethyl ether, frozen and lyophilized. The water extract afforded10 mg of material and the acetic acid extract afforded 43 mg (58%total). The two fractions were combined and purified by preparative HPLC(reverse phase, 50 mL/minute, 10-60% acetonitrile/water with 0.1%trifluoracetic acid, 40 minute program). Acetonitrile was removed undervacuum from the appropriate fraction. The remaining liquid was frozenand lyophilized to afford the above-identified compound as a whitefluffy powder. Mass spectral analysis showed the expected molecular ionat 661.3 (calc. 661.3).

Example 15 Preparation of ##STR45##

A 0.637 g portion of the compound of Example 6 (1.15 mmole) wasconverted to the above-identified product using the methods described inExample 9 and using 2-propylpentanoic acid (in place of 4-methylvalericacid). Flash chromatography (silica, 10% methanol/dichloromethane)afforded 0.547 g (60%) of the above-identified compound as a foam. R_(f)=0.33 (10% methanol/dichloromethane).

Example 16 Preparation of ##STR46##

A 0.505 g portion of the compound of Example 15 (0.646 mmole) wasoxidized and worked up as described in Example 7. Flash chromatography(silica, 4:1:4 hexane/methanol/dichloromethane) afforded 0.467 g (95%)of the above-identified compound as a foam. R_(f) =0.38 (10%methanol/dichloromethane).

Example 17 Preparation of ##STR47##

A 0.105 g portion of the compound of Example 16 (0.134 mmole) wasdeprotected using hydrofluoric acid as described in Example 15 to afford30 mg (43%) of material. This material was purified by preparative HPLC(reverse phase, 50 mL/minute, 10-50% acetonitrile/water with 0.1%trifluoroacetic acid, 40 minute program). Acetonitrile was removed undervacuum from the appropriate fraction. The remaining liquid was frozenand lyophilized to afford the above-identified compound as a whitefluffy powder. Mass spectral analysis showed the desired ion at 643.3(calc. 643.4).

Example 18 Preparation of ##STR48##

A 0.89 g portion of the compound of Example 3 (2.45 mmole) was convertedto the amide as described in Example 5, using 3-phenylpropylamine (inplace of 2-phenylethylamine). Flash chromatography (silica, 4:1:4hexane/methanol/dichloromethane) afforded 1.03 g (90%) of theabove-identified compound as a foam.

Example 19 Preparation of ##STR49##

A 0.5 g portion of the compound of Example 18 (1.07 mmole) was convertedto the above product as described in Example 6. Flash chromatography(silica, 10% methanol/dichloromethane) afforded 0.735 g (89%) of theabove-identified compound as a foam. R_(f) =0.27 (10%methanol/dichloromethane).

Example 20 Preparation of ##STR50##

A 0.70 g portion of the compound of Example 19 (0.909 mmole) wasoxidized and worked up as described in Example 7. Flash chromatography(silica, 4:1:4 hexane/methanol/dichloromethane) afforded 0.612 g (87%)of the above-identified compound as a foam. R_(f) =0.41 (10%methanol/dichloromethane).

Example 21 Preparation of ##STR51##

A 0.512 g portion of the compound of Example 20 (0.667 mmole) washydrogenated and worked up as described in Example 8. The resulting foamwas purified by preparative HPLC (reverse phase, 50 mL/minute, 20-60%acetonitrile/water with 0.1% trifluoroacetic acid, 40 minute program)The appropriate fractions were combined, the acetonitrile was removedunder vacuum. The remaining liquid was frozen and lyophilized to afford0.177 g (42%) of the above-identified compound as a white fluffy powder.Mass spectral analysis showed the expected molecular ion at 631.3 (calc.631.3).

Example 22 Preparation of L-proline-9-fluorenemethyl esterm-toluenesulfonic acid salt ##STR52##

A solution of L-proline (15.99 g, 139.0 mmole), 9-fluorenemethanol (30.0g, 152.9 mmole), and p-toluenesulfonic acid in 600 mL of toluene wasrefluxed and water was removed with a Dean-Stark trap. After 26 hours,the reaction was concentrated to give 64 g (99% crude yield) of theabove-identified compound as an oil which was used directly in the nextstep.

Example 23 Preparation of α-N-(t-butoxycarbonyl)-L-aspartyl-(β-benzylester)-L-proline-9-fluorenemethyl ester ##STR53##

A solution of L-proline-9-fluorenemethyl ester p-toluenesulfonic acidsalt (the product of Example 22) (15.44 g, 33.2 mmole),α-N-(t-butoxycarbonyl)-L-aspartic acid-(β-benzyl ester) (9.35 g, 41.9mmole),benzotriazol-1-yloxy-tris-(dimethylamino)-phosponium-hexafluorophosphate(18.6 g, 42.0 mmole) in 100 mL dimethylformamide was allowed to stir inan ice-bath. This solution was treated with 1-HOBt hydrate (0.45 g, 3.34mmole), diisopropylethylamine (19.0 mL, 198 mmole) and the reactionallowed to stir at about 0 to 5° C. for 1.5 hours. After this time thereaction mix was poured into 600 mL of ethyl acetate and extractedsuccessively with saturated aqueous citric acid, water, saturated sodiumbicarbonate, and finally brine. The organic phase was dried withanhydrous magnesium sulfate and concentrated under vacuum to give 18 g(91% crude yield) of an oil, which was used directly in the next step.

Example 24 Preparation of α-N-(t-butoxycarbonyl)-L-aspartyl-(β-benzylester)-L-proline ##STR54##

The crude oil from Example 23,α-N-(t-butoxycarbonyl)-L-aspartyl-(β-benzyl ester)-L-proline9-fluorenemethyl ester (17.5 g, 29.2 mmole), was suspended in 250 mLtriethylamine and allowed to reflux for 1 hour. This mixture wasconcentrated to an oil, dissolved in 600 mL of ethyl acetate. The ethylacetate phase was washed once with a citric acid solution, once withbrine, dried with anhydrous magnesium sulfate, and concentrated undervacuum to give an oil. This material was purified by columnchromatography (silica gel, 10-20% tetrahydrofuran/dichloromethane) togive 7.5 g (yield about 38% overall) of the above-identified compound.

Example 25 Preparation of ##STR55##

A solution of 400 mg of the compound of Example 4 (1.14 mmole) wasdissolved with stirring into 2 mL of dimethylformamide. This solutionwas treated successively with D-phenylalanine benzyl esterp-toluenesulfonic acid salt (489 mg, 1.14 mmole), NMM (0.342 mL, 3.11mmole) and BOP (5.15 mg, 1.16 mmole). After 2 hours, thin layerchromatographic analysis (10% methanol/dichloromethane) showed no morematerial corresponding to the acid. The reaction mixture was poured intoethyl acetate (300 mL) and washed successively with 1N aqueoushydrochloric acid (75 mL), water (75 mL), saturated sodium bicarbonate(75 mL) and brine (75 mL). The organic layer was dried over anhydrousmagnesium sulfate and concentrated under vacuum. This afforded 600 mg(90%) of the above-identified compound as a white foam. R_(f) =0.70 (twospots, 10% methanol/dichloromethane).

Example 26 Preparation of ##STR56##

To a solution of 586 mg of the compound of Example 25 (1.00 mmole) in 17mL dichloromethane, was added 17 mL of trifluoroacetic acid withstirring. After 30 minutes, thin layer chromatographic analysis (10%methanol/dichloromethane) showed no starting material. Thetrifluoroacetate salt was isolated by concentrating the solution. Theresidue was dissolved in toluene and then concentrated to an oil, whichhad some trifluoroacetic acid. The oil was dissolved in 3 mL ofdimethylformamide with stirring and this solution treated with (264 mg,1.49 mmole) α-N-t-butoxycarbonyl-L-proline, 0.600 mL (6.7 mmole) NMM and554 mg (1.23 mmole) BOP. After 1 hour, thin layer chromatographicanalysis (10% methanol/dichloromethane) showed no more trifluoroacetatesalt. The mixture was added to 300 mL of ethyl acetate and washedsuccessively with 75 mL of 1N aqueous hydrochloric acid, 75 mL of water,75 mL of saturated sodium bicarbonate and 75 mL of brine. The organiclayer was dried over anhydrous magnesium sulfate and concentrated undervacuum. This afforded 700 mg of the above-identified compound as a whitefoam, R_(f) =0.70 (two spots, 10% methanol/dichloromethane).

Example 27 Preparation of ##STR57##

This compound was synthesized by the procedure described in Example 26,except that an equimolar amount of the compound of Example 26 andBoc-L-aspartic acid-(β-benzyl ester) was used, instead ofα-N-t-butoxycarbonyl-L-proline. This gave 825 mg of the above-identifiedcompound (R_(f) =0.5, two spots, 10% methanol/dichloromethane).

Example 28 Preparation of ##STR58##

A 370 mg portion of the compound of Example 27 (0.42 mmole) wasdissolved in 4 mL of dimethylsulfoxide with stirring. This solution wastreated with 4 mL of toluene and 816 mg (4.26 mmole) ofethyl-3-(3-dimethylamino)propylcarbodiimide hydrochloride followed by0.150 mL of dichloroacetic acid. After 1 hour, thin layerchromatographic analysis (10% methanol/dichloromethane) showed a singlenew spot and no starting material. The mixture was added to 500 mL ofethyl acetate and washed with two 200 mL portions of water and 150 mL ofbrine. The organic layer was dried over anhydrous magnesium sulfate andconcentrated under vacuum. Flash chromatography (silica, 0 to 4%methanol/dichloromethane) afforded 200 mg (54% yield) of theabove-identified compound as a foam. R_(f) =0.55 (10%methanol/dichloromethane).

Example 29 Preparation of ##STR59##

A 0.160 mg portion of the compound of Example 28 (0.180 mmole) wasdissolved in 25 mL of methanol. This solution was added to a Parr vesselcontaining 150 mg 10% Pd/C, followed by 0.20 mL (0.20 mmole) 1N aqueoushydrochloric acid and 0.2 mL of glacial acetic acid. The mixture wasshaken under a 10 psig hydrogen atmosphere for 1.5 hours, after whichHPLC (reverse phase, 1 mL/minute, 5-95% acetonitrile/water with 0.1%trifluoroacetic acid, 20 minute program, retention time=14.5 minute)showed complete reaction. The mixture was filtered, rinsed with 10 mL ofmethanol and concentrated under vacuum. The resulting foam was purifiedby preparative HPLC (reverse phase, 50 mL/minute, 10-60%acetonitrile/water with 0.1% trifluoroacetic acid, 40 minute program).The appropriate fractions were combined and the acetonitrile was removedunder vacuum. The remaining liquid was frozen and lyophilized to afford100 mg of the above-identified compound as a white fluffy powder. Massspectral analysis showed the expected molecular ion at 661.3 (calc.661.3).

Example 30 Preparation of ##STR60##

To a solution of t-butoxycarbonylmethioninesulfone acid (14.0 g, 50.0mmole) in dichloromethane (150 mL) at 0° C. was added HOBt (10.1 g, 75mmole) followed by dicyclohexylcarbodiimide (11.33 g, 55.0 mmole). Themixture was stirred for 10 minutes, and then proline benzyl esterhydrochloride salt (50.0 mmole, 12.0 g) was added followed by NMM (100mmole, 10.9 mL). The resulting mixture was stirred in an ice bath andallowed to come to room temperature over 12 hours. The mixture was thenfiltered to remove dicyclohexylurea and ethyl acetate (300 mL) is added.The organic phase was then added to a separatory funnel and washed withsaturated aqueous sodium bicarbonate, brine and then 1M aqueous HCl. Theorganic phase was dried over magnesium sulfate and then filtered. Theorganic phase was then reduced on a rotary evaporator in vacuo and thenon a high vacuum line to remove traces of solvent to provide 23.5 g of awhite solid (100%). Rf=0.34 (silica gel, trichloromethane/methanol(95:5)).

Example 31 Preparation of ##STR61##

To a solution of t-butoxycarbonylmethioninesulfoneproline benzylester(23.5 g, 50 mmole) in dry dioxane (300 mL) was added 100 mL of a 4M HCldioxane solution. The mixture was then stirred at room temperature for 1hour until the starting material disappeared as shown by thin layerchromatography analysis (10% trichloromethane:methanol). The diethylether was added to the mixture to precipitate the white hydrochloridesalt. The mixture was filtered on a Buchner funnel and then dried underhigh vacuum to provide 20.16 g (100%) of a white solid.

Example 32 Preparation of ##STR62##

To a solution of methioninesulfoneproline benzylester hydrochloride(20.0 mmole, 8.08 g) in dry acetonitrile (100 mL) cooled to 0° C. wasadded α-toluenesulfonylchloride (20.0 mmole, 3.8 g) all at once followedby pyridine (50.0 mmole, 4.2 mL). The mixture was then stirred in theice bath for 12 hours eventually warming to room temperature. Work-upconsisted of reducing the volume in vacuo and diluting with ethylacetate (300 mL). The organic phase was then washed with saturatedaqueous sodium bicarbonate, brine and 1M aqueous HCl (100 mL). Theorganic phase was dried over magnesium sulfate, filtered and evaporatedin vacuo to provide 8.8 g (100%) of a foamy golden solid. Rf=0.31(silica gel, trichloromethane:methanol (95:5)). The solid was filteredthrough a plug of silicon dioxide (50 g) using ethylacetate beforehydrogenation to eliminate possible sulfur related impurities.

Example 33 Preparation of ##STR63##

To a solution of α-toluenesulfonylmethioninesulfoneproline benzylester(8.8 g, 20 mmole) in methanol (300 mL) was added 1.0 g of 10% Pd/C. Themixture was then hydrogenated at 1 atmosphere of hydrogen gas and roomtemperature. The mixture was stirred for 12 hours. The mixture was thenfiltered and the organic phase reduced in vacuo to provide 8.0 g (100%)of a white foamy solid.

Example 34 Preparation of ##STR64##

A 600 mg (1.1 mmole) portion of the product of Example 5 was taken up intrifluoroacetic acid at 0° C., and stirred for two hours. This solutionwas diluted with toluene (100 mL) and reduced in vacuo. This residue wasdissolved in dimethylformamide (6 mL) and 361 mg (1.1 mmole) ofα-toluenesulfonylglycineproline were added followed by 538 mg (1.22mmole) of BOP and 1117 mg (11.0 mmole, 1.21 mL) of NMM and the solutionwas allowed to stir overnight.

This solution was diluted in 50 mL 1M HCl and extracted three times withethyl acetate. The organics were combined and washed with water (threetimes), saturated sodium bicarbonate and brine. The solution was driedover magnesium sulfate and concentrated in vacuo to give 405 mg of theabove compound as an orange/yellow foam. Thin layer chromatographyshowed no more starting material.

Example 35 Preparation of ##STR65##

A 400 mg (0.605 mmole) portion of the product of the previous example,was taken up in 10 mL of 1:1 toluene:DMSO with EDC. To this solution wasadded 312 mg (0.2 mL, 2.42 mmole, 4.0 eq) of dichloroacetic acid. Thissolution was stirred for one hour and ten minutes, diluted with 50 mLwater and extracted twice with ethyl acetate (100 mL). The organics werecombined, washed with brine, dried over magnesium sulfate, andconcentrated in vacuo. This solution was purified on a (4:1:4hexanes:methanol:dichlormethane) silica column to give 150 mg of a(clean) white solid.

Example 36 Preparation of ##STR66##

The product of the previous example was subjected to hydrogen fluorideas previously described and purified by HPLC to give the above compoundwhich had an actual mass spectra peak (613.2) that correlated well withthe expected value of 613.3.

Example 37 Preparation of ##STR67##

To cyclohexylamine sulfamic acid sodium salt (Aldrich, 2.01 g, 10.0mmole) was added 6 mL phosphorousoxychloride. The white suspension wasthen heated to 100° C. for 4 hours. The mixture was then cooled to roomtemperature and the phosphorousoxychloride was stripped off in vacuo toprovide a white solid. This solid was then suspended in dry acetonitrile(35 mL) and then cooled to 0° C. To this mixture was added themethionine sulfone benzyl ester hydrochloride salt of Example 31 (3.07g, 10.0 mmole) followed by pyridine (2.6 mL, 30.0 mmole). The mixturewas allowed to warm to room temperature in the ice bath over 10 hours.The acetonitrile was stripped off in vacuo and then diluted with ethylacetate. The organic phase was washed with saturated aqueous sodiumbicarbonate, brine and 1M aqueous HCl. The organic phase was dried overmagnesium sulfate, filtered and reduced in vacuo to provide 4.8 g of acrude yellow solid. The solid was washed with ethyl ether and thenfiltered to provide 3.8 g (88%) of an off white solid.

Example 38 Preparation of ##STR68##

The product of the previous example was dissolved in 100 mL of a 1:1mixture of tetrahydrofuran/methanol and 0.5 g of 10% Pd/C was added. Themixture was hydrogenated at 1-atmosphere of hydrogen for 4 hours at roomtemperature. The mixture was then filtered and reduced in vacuo toprovide 3.2 g of a white solid.

Example 39 Preparation of ##STR69##

To a solution of the cyclohexylsulfonylureamethioninesulfone acidproduct of Example 33 (2.75 g, 8.0 mmole) in dry dimethylformamide (10mL) was added EDC (8.0 mmole, 1.53 g) and HOBt (12 mmole, 1.62 g) all atonce. This mixture was stirred at 0° C for 10 minutes, then prolinebenzylester hydrochloride (8 mmole, 1.93 g) was added followed by NMM(24 mmole, 2.6 mL). The reaction was allowed to come to room temperaturein an ice bath over 10 hours. The reaction mixture was then diluted withethyl acetate and washed with saturated aqueous sodium bicarbonate,brine and 1M aqueous HCl. The organic phase was dried over magnesiumsulfate, filtered and reduced in vacuo to provide 3.25 g (75%) of aviscous foamy solid. Rf=0.19 (silica gel, trichloromethane:methanol(95:5)).

Example 40 Preparation of ##STR70##

To a solution of the benzyl ester product of Example 39 (3.2 g, 6.0mmole) in methanol (60 mL) was added 15 mL of a 2.0M lithium hydroxidesolution at room temperature. The clear solution was stirred for 1 hourand then the methanol was stripped off in vacuo. The aqueous solutionwas then washed with ethyl ether (2×100 mL) and then the aqueoussolution was neutralized to pH 1 with 1M aqueous HCl and extracted twicewith 100 mL ethyl acetate. The organic phase was then washed with brine,dried over magnesium sulfate, filtered and reduced in vacuo to provide2.3 g (90%) of a white fluffy solid. Rf=0.13 (silica gel,trichloromethane:methanol (70:30)).

Example 41 Preparation of ##STR71##

The product of the previous example and the product of Example 5 werecoupled as described in various examples herein.

Example 42 Preparation of ##STR72##

The product of the previous example was oxidized and worked up asdescribed in Example 35.

Example 43 Preparation of ##STR73##

The product of the previous example was subjected to hydrogen fluorideas previously described and purified by HPLC to give the above compound.

Example 44 Preparation of ##STR74##

To methyl sulfate sodium salt (Aldrich, 1.34 g, 10.0 mmole) is added 10mL phosphorousoxychloride and the mixture heated to 100° C. for 3 hours.The reaction is cooled to room temperature and thephosphorousoxychloride removed in vacuo to leave a white residue. Theresidue is then mixed with dry acetonitrile (25 mL) and cooled to 0° C.in an ice bath. Then the methioninesulfoneproline benzylesterhydrochloride salt (4.04 g, 10 mmole) is added all at once followed bypyridine (2.6 mL, 30 mmole). The reaction is allowed to warm to roomtemperature in the ice bath over 10 hours. The acetonitrile is removedin vacuo and the residue diluted with ethyl acetate. The organic phaseis washed with saturated aqueous sodium bicarbonate, brine and 1Maqueous HCl. The organic phase is dried over magnesium sulfate, filteredand the solvent removed in vacuo to provide the coupled product in ayield of 4.6 g (100%).

Example 45 Preparation of ##STR75##

The product of the previous example is dissolved in 150 mL of methanoland 0.5 g of 10% Pd/C is added. The mixture is then hydrogenated atatmospheric pressure for 4 hours. The mixture is filtered and thesolvent removed in vacuo to provide the corresponding acid in a yield of3.6 g (100%).

Example 46 Preparation of ##STR76##

The product of Example 5 and the product of the previous example arecoupled as described in various examples herein.

Example 47 Preparation of ##STR77##

The product of the previous example was oxidized and worked up asdescribed in Example 35.

Example 48 Preparation of ##STR78##

The product of the previous example is subjected to hydrogen fluoride aspreviously described and purified by HPLC to give the above compound.

Example 49 Preparation of ##STR79##

To 3-(trimethylsilyl)-1-propanesulfonic acid sodium salt (available fromAldrich or Huls America) (5.0 g, 23.0 mmole) was added 10 mLphosphorousoxychloride . The mixture was then heated to 100° C. for 3hours and then cooled to room temperature. The phosphorousoxychloridewas then removed in vacuo and the residue partitioned betweenethylacetate and ice. After the ice melted the ethyl acetate phase wasseparated and washed three times with saturated aqueous sodiumbicarbonate until pH 8 by litmus paper was observed. The ethyl acetatewas then washed with brine and dried over magnesium sulfate, filteredand the solvent removed in vacuo to provide 4.04 g (82%) of a yellowishoil. Rf=0.52 (silica gel; hexanes:ethyl acetate (90:10)).

To a solution of the above 3-(trimethylsilyl)-1-propanesulfonyl chloride(2.14 g, 10.0 mmole) in dry acetonitrile (20 mL) cooled to 0° C. in anice bath was added the methioninesulfoneproline benzylesterhydrochloride (4.04 g, 10.0 mmole) followed by pyridine (2.55 mL, 30.0mmole). The mixture was allowed to warm to room temperature in the icebath over the course of 10 hours. The acetonitrile was then stripped offin vacuo and the residue diluted with ethyl acetate. The organic phasewas washed with saturated aqueous Sodium bicarbonate, brine and 1Maqueous HCl. The organic phase was then dried over magnesium sulfate,filtered and the solvent removed in vacuo to provide 4.85 g (89%) of aviscous oil. Rf=0.23 (silica gel, trichloromethane:methanol (95:5)).

Example 50 Preparation of ##STR80##

To a solution of 3-(trimethylsilyl)-1-propanesulfonyl methionine sulfoneproline benzylester (4.8 g,8.8 mmole) in methanol (150 mL) was added 25mL of a 2.0M lithium hydroxide solution. The mixture was stirred at roomtemperature for 1 hour and then the methanol was removed in vacuo. Theaqueous phase was washed twice with 100 mL ethyl ether and thenneutralized to pH 1 with 1M aqueous HCl. The aqueous phase wasextracted, filtered, and the solvent removed in vacuo to provide 3.24 g(81%) of the corresponding acid. R=0.25 (silica gel,trichloromethane:methanol (70:30)).

Example 51 Preparation of ##STR81##

The product of Example 5 and the product of the previous example werecoupled as described in various examples herein.

Example 52 Preparation of ##STR82##

The product of the previous example was oxidized and worked up using theprocedure of Example 74.

Example 53 Preparation of ##STR83##

To a solution of the compound of the previous example (8.8 g, 20 mmole)in methanol (300 mL) was added 1.0 g of 10% Pd/C. The mixture was thenhydrogenated at 1 atmosphere and room temperature. The mixture wasstirred for 12 hours. The mixture was then filtered and the organicphase reduced in vacuo to provide 8.0 g (100%) of a white foamy solid.

Example 54 Preparation of ##STR84##

A 6.0 g (12.4 mmole) portion of the product of methioninesulfoneprolinebenzylester trifluoroacetic acid salt was reacted with 2.07 mL (16mmole) of n-butanesulfonylchloride and 5.0 mL (36 mmole) oftriethylamine in dichloromethane from 0° C. to room temperature. Thereaction mixture was poured into saturated aqueous bicarbonate andextracted with ethyl acetate (2×100 mL). The organic phase was washedwith brine and 1M aqueous HCl. The organic phase was separated and driedover magnesium sulfate, filtered and reduced in vacuo to give 5.73 g ofviscous oil. The oil was mixed with 2M potassium hydroxide (20 mL) and100 mL methanol at room temperature for two hours. The methanol wasreduced in vacuo and the aqueous solution was then washed with ether(2×50 mL) and then neutralized with 1M HCl to a pH of 1, The aqueoussolution was then extracted with ethyl acetate (2×100 mL) and dried overmagnesium sulfate, filtered and reduced in vacuo to give 2.85 g of theabove acid as a viscous foamy solid. The overall yield was 60.5%.

A 500 mg (1.1 mmole) portion of the product of Example 5 was taken up intrifluoroacetic acid at 0° C. and stirred for two hours. This mixturewas diluted with toluene (100 mL) and concentrated in vacuo two times.The residue was taken up in dimethylformamide. To this solution wereadded 440 mg (1.1 mmole, 1.0 equivalents) of the above acid of theprevious paragraph, 1.21 mL (1117 mg, 11.0 mmole, 10.0 equivalents) ofNMM and 538 mg (1.22 mmole, 1.1 equivalents) of BOP. This solution wasstirred overnight. The solution was diluted in 120 mL of 1M HCl andextracted three times with 50 mL of ethyl acetate. The organic phaseswere combined, washed with 1M HCl, water (three times), saturated sodiumbicarbonate and brine and then dried over magnesium sulfate. It wasconcentrated in vacuo to give 380 mg of the above crude product.

Example 55 Preparation of ##STR85##

The product of the previous example was taken up in 10 mL of 1:1PheMe:DMSO and 994 mg (5.19 mmole, 10.0 equivalents) of EDC and 0.17 mL(267 mg, 2.07 mmole, 4.0 equivalents) dichloroacetic acid were added.The reaction mixture was allowed to stir for about one hour and tenminutes and then diluted with water (about 50 mL). It was extractedtwice with ethyl acetate. The organic phases were combined, washed withbrine, dried over magnesium sulfate and concentrated in vacuo. Theconcentrate was purified on a (4:1:4 hexanes:methanol:dichloromethane)silica column, to give 80 mg of the above compound.

Example 56 Production of ##STR86##

The product of the previous example was cleaved with hydrogen fluorideand purified by HPLC to give the above product with an observed massspectra peak of 685.3 that agreed exactly with the expected value.

Example 57 Preparation of ##STR87##

A 10 g (27.5 mmole, 1.0 equivalent) portion of the product of Example 5was taken up in methanol and 34.4 mL (34.4 mmole, 1.25 equivalents) oflithium hydroxide were added. This solution was allowed to stirovernight. The solution was concentrated to 90 mL, in vacuo diluted with500 mL of water, and extracted three times with ethyl acetate. Theaqueous phase was then concentrated to about 400 mL, and gravityfiltered through Dowex 50 resin (150 mL bed in a sintered glass funnel)

The resin was washed with 800 mL of water and 500 mL of 50:50methanol/water until no more UV active resin material was observed. Thematerial was concentrated in vacuo, and reconcentrated twice withacetonitrile. Then 2.0 g (5.71 mmole) of this material was taken up in28.5 mL of dimethylformamide.

To this solution were added 0.663 mL (500 mg, 5.71 mmole, 1.0equivalents) of isoamylamine, 1.88 mL (1733 mg, 17.13 mmole, 3.0equivalents) of NMM, and 2778 mg (6.28 mmole, 1.1 equivalents) of BOP.This solution was allowed to stir over 48 hours.

This solution was diluted in 1M HCl and extracted three times with ethylacetate. The organics were recombined, washed with 1M HCl (once), water(three times), saturated sodium bicarbonate, and brine. The solution wasthen dried over magnesium sulfate and concentrated in vacuo to 1.5 g ofthe above compound.

Example 58 Production of ##STR88##

The product of the previous example was reacted with the product ofExample 33 using the procedure of Example 34 to give the above compound.

Example 59 Production of ##STR89##

The product of the previous example was oxidized and worked up to givethe above compound.

Example 60 Production of ##STR90##

The product of the previous example was cleaved with hydrogen fluorideto give the above compound.

Example 61 Preparation of ##STR91##

A 3 g (6.878 mmole) portion of the product of Example was added to 69 mLof acetonitrile. To this mixture was added 2.339 g (10.317 mmole, 1.5eq) of 2-naphthylsulfonylchloride and 4.201 g (4.115 mL, 34.39 mmole, 5eq) of pyridine and stirred for 10 hours. This mixture was concentratedin vacuo and diluted with ethyl acetate (500 mL) followed by washingwith 1M HCl, water, aqueous sodium bicarbonate, and brine. The organicphase was dried over magnesium sulfate and concentrated in vacuo. Thinlayer chromatography (10% methanol/dichloromethane) showed some2-naphthylsulfonylchloride. The mixture was then filtered (silica,dichloromethane (100 mL) then 10% methanol/dichloromethane (200 mL) togive 3.96 g of the above compound

Example 62 Preparation of ##STR92##

A 3.96 g (7.088 mmole) portion of the above product was dissolved in 250mL of methanol with a trace of tetrahydrofuran. To this solution 2 g of10% Pd/C was added under nitrogen and stirred under hydrogen at oneatmosphere of pressure. Thin layer chromatography (10%methanol/dichloromethane) showed no starting material. This solution wasthen filtered through a nylon filter and concentrated in vacuo to givethe above compound. Yield was 3.2 g (96%).

Example 63 Preparation of ##STR93##

A 0.603 g (1.288 mmole) portion of the product of the previous exampleand a 0.5 g (1.288 mmole) portion ofnitroarginine-α-hydroxy-2-phenylethylamide hydrochloride salt weredissolved in 13 mL of dimethylformamide with stirring. To this solutionwas added 0.651 g (0.708 mL, 6.44 mmole) of NMM and 566 mg BOP and thereaction stirred hours. This solution was extracted with ethyl acetate(600 mL), 200 mL of water, 200 mL of 1M HCl, 200 mL of water, 200 mL ofsodium bicarbonate and 200 mL of brine; it was dried over magnesiumsulfate and concentrated in vacuo. Thin layer chromatography (10%methanol/dichloromethane) showed no starting material. The abovecompound was obtained in a yield of 0.71 g (71%).

Example 64 Preparation of ##STR94##

The 0.71 g of the product of the previous example was dissolved in 18 mLof 1:1 solution of toluene/DMSO with stirring. To this solution wereadded 1.695 g (8.84 mmole) of EDC and 0.456 g (0.292 mL, 3.536 mmole) ofdichloroacetic acid. After 1.5 hours, thin layer chromatography (10%methanol/dichloromethane) showed no starting material. This solution wasextracted with 500 mL of ethyl acetate, 200 mL of water, 200 mL ofsodium bicarbonate and 200 mL of brine; it was dried over magnesiumsulfate and concentrated in vacuo. Purification on a silica column usinga 4:1:4 hexanesmethanoldichloromethane as eluent gave 0.688 g (0.884mmole) of the above compound.

Example 65 Preparation of ##STR95##

The product of the previous example was subjected to hydrogen fluorideas previously described and purified by HPLC to give the above compound.

Example 66 Preparation of ##STR96##

A 5 g (20.071 mmole) portion of t-butoxycarbonylmethioninesulfone acidwas dissolved in 80 mL of dimethylformamide with stirring. To thissolution were added 7.898 g (20.071 mmole) of isoleucine benzylesterpara-toluenesulfonic acid salt, 6.091 g (6.621 mL, 60.213 mmole, 3equivalents) of NMM and 8.877 g (20.071 mmole, 1 equivalent) of BOP.This solution was extracted with 600 mL of ethyl acetate, 200 mL ofwater, 200 mL of HCl, 200 mL of water, 200 mL of aqueous sodiumbicarbonate, and 200 mL of brine; it was dried over magnesium sulfateand concentrated in vacuo. Thin layer chromatography (10%methanol/dichloromethane showed no more starting material. To thissolution was added 100 mL of 4M HCl/dioxane. After approximately fivehours thin layer chromatography showed no starting material. Thesolution was concentrated to give methioninesulfoneisoleucinebenzylester hydrochloride in a yield of 8.9 g (98%).

A 2.37 g (5.641 mmole) portion of the above salt and a 1.613 g (8.462mmole, 1.5 equivalent) portion of α-toluenesulfonylchloride were mixedwith stirring in acetonitrile. To this solution was added 3.446 g(28.205 mmole, 5 equivalent) portion of pyridine and the reactionstirred for 10 hours. This solution was concentrated, in vacuo andextracted with 600 mL of ethyl acetate, 100 mL of water, 100 mL of HCl,100 mL of water, 100 mL of aqueous sodium bicarbonate and 100 mL ofbrine. This solution was then dried, filtered and concentrated. Thinlayer chromatography (10% methanol/dichloromethane) showed a secondspot. The solution was filtered (silica, dichloromethane--10% (100 mL)then methanol/dichloromethane (200 mL)). This gave compound 80 in ayield of 7.87 g (95%).

Example 67 Preparation of ##STR97##

The 2.87 g (5.333 mmole) of the product of the previous example wasdissolved in 150 mL of methanol and 100 mL of tetrahydrofuran withstirring. The solution was purged with nitrogen, and 1.5 g of Pd/C wasadded and stirred under atmosphere of hydrogen overnight. The solutionwas filtered, and concentrated. This gave the above compound in a yieldof 0.46 g (20%).

Example 68 Preparation of ##STR98##

A 0.5 g (1.288 mmole) portion of the nitroarginine-αhydroxy-2-phenylethylamide hydrochloride salt and a 0.577 g (1.288mmole) portion of the product of the previous example were dissolved in6 mL of dimethylformamide with stirring. To this solution were added0.651 g (6.44 mmole, 5 equivalents) of NMM and 0.57 g (1.288 mmole, 1equivalent) of BOP and the reaction stirred for 10 hours. This solutionwas extracted with 500 mL of ethyl acetate, 100 mL of water, 100 mL ofHCl, 100 mL of water, 100 mL of aqueous sodium bicarbonate, and 100 mLof brine; it was dried over magnesium sulfate and concentrated. Thinlayer chromatography (10% methanol/dichloromethane) showed a minorcontaminant. This gave the above compound in a yield of 0.541 g (54%).

Example 69 Preparation of ##STR99##

The 0.541 g (0.691 mmole) of the product of the previous example wasdissolved in 14 mL of 1:1 toluene/DMSO with stirring. To this solutionwere added 1.325 g (6.91 mmole, 10 equivalents) of EDC and 0.356 g(0.225 mL, 2.764 mmole, 4 equivalents) of dichloroacetic acid. After onehour thin layer chromatography (10% methanol/dichloromethane) showed nostarting material.

The solution was extracted with 300 mL of ethyl acetate, 200 mL ofwater, 200 mL of water, 150 mL of aqueous sodium bicarbonate and 150 mLof brine; it was dried over magnesium sulfate and concentrated. Thisgave the above compound in a yield of 80 mg (15%).

Example 70 Preparation of ##STR100##

The product of the previous example was treated with hydrogen fluorideas in Example 14 and then purified using HPLC to give the abovecompound.

Example 71 Preparation of ##STR101##

To a suspension of glycine ethyl ester hydrochloride (8.0 Hg, 60.0mmole) in dry hydrogen fluoride (150 mL) cooled to 0° C. in an ice bathwas added the α-toluenesulfonyl chloride (9.5 g, 50.0 mmole) followed bypyridine (12.1 mL, 150 mmole). The reaction was stirred in the ice bathover 10 h allowing the reaction to warm to room temperature. Thehydrogen fluoride was then removed in vacuo and the resulting residuediluted with ethylacetate. The organic solution was then washed withsaturated aqueous sodium bicarbonate, brine then 1M aqueous HCl. Theorganic phase was dried (MgSO₄), filtered and then reduced in vacuo toprovide 6.67 g reddish oil (52%). Rf=0.83 (3:2, Hexanes:Ethyl Acetate).

The above oil was diluted with MeOH (70 mL) and 30 mL of a 1.5M LiOHaqueous solution was added. The mixture was stirred at 25° C. for 1hour. The MeOH was then reduced in vacuo. to provide 3.14 g (53%) of atan solid. Rf=0.25 (70:30; CHCl₃ :MEOH).

Example 72 Preparation of ##STR102##

To a solution of the α-Toluenesulfonylglycine acid (1.5 g, 6.5 mmole) inCH₂ Cl₂ (25 mL) cooled to 0° C. was added HOBt (1.3 g, 9.8 mmole)followed by pCC (1.5 g, 7.2 mmole). This mixture is stirred for 10minutes at which time prolinebenzyl hydrochloride (1.74 g, 7.2 mmole)was added followed by NMM (1.1 mL; 10 mmole). The mixture was thenstirred in the ice bath over 10 h eventually coming to room temperature.The mixture was then filtered through a Buchner funnel and the organicphase was diluted with ethyl acetate and washed with saturated aqueoussodium bicarbonate, brine and 1M aqueous HCl. The organic phase wasdried (MgSO₄), filtered and reduced in vacuo to provide 2.8 g of aviscous oil (100%). R_(f) =0.5 (95:5, CHCl₃ ; MeOH).

Example 73 Preparation of ##STR103##

The above oil was diluted with MeOH (70 mL) and 30 mL of a 1.5M LiOHaqueous solution was added. The mixture was stirred at 25° C. for 1hour. The MeOH was then reduced in vacuo and the residue partitionedbetween ethyl ether and water. The aqueous phase was washed with ether(2×100 mL) and then the aqueous phase was neutralized to pH 1 with 1Maqueous HCl. The aqueous phase was then extracted with ethyl acetate(2×100 mL). The organic phase was then dried (MgSO₄), filtered andreduced in vacuo to provide 3.14 g (53%) of a tan solid. Rf=0.25 (70:30;trichloromethane:methanol).

Example 74 Preparation of ##STR104##

A 600 mg (1.1 mmole) portion of the product of Example 5 was taken up intrifluoroacetic acid at 0° C., and stirred for two hours. This solutionwas diluted with toluene (100 mL) and reduced in vacuo. This residue wasdissolved in dimethylformamide (6 mL) and 361 mg (1.1 mmole) of theα-toluenesulfonylglycineproline acid of the previous example were addedfollowed by 538 mg (1.22 mmole) of BOP and 1117 mg (11.0 mmole, 1.21 mL)of NMM and the solution was allowed to stir overnight.

This solution was diluted in 50 mL 1M HCl and extracted three times withethyl acetate. The organics were combined and washed with water (threetimes), saturated sodium bicarbonate and brine. The solution was driedover magnesium sulfate and concentrated in vacuo to give 405 mg of theabove compound as an orange/yellow foam. Thin layer chromatographyshowed no more starting material.

Example 75 Preparation of ##STR105##

A 400 mg (0.605 mmole) portion of the product of the previous example,was taken up in 10 mL 1:1 toluene:DMSO with EDC. To this solution wasadded 312 mg (0.2 mL, 2.42 mmole, 4.0 eq) of dichloroacetic acid. Thissolution was stirred for one hour and ten minutes, diluted with 50 mLwater and extracted twice with ethyl acetate (100 mL). The organics werecombined, washed with brine, dried over magnesium sulfate, andconcentrated in vacuo. This solution was purified on a (4:1:4hexanes:methanoldichlormethane) silica column to give 150 mg of theabove compound as a clean white solid.

Example 76 Preparation of ##STR106##

The product of the previous example was cleaved with hydrogen fluoride.The product was purified by HPLC to give the above compound which had anactual mass spectra peak (613.2) that correlated well with the expectedvalue of 613.3.

Example 77 Preparation of ##STR107##

A 9.0 g (40 mmole) portion of 2-naphthylsulfonylchloride, a 5.6 g (40mmole) portion of glycineethylesterhydrochloride salt, and a 14 mL (100mmole) portion of triethylamine were mixed in tetrahydrofuran at 0° C.to give a yield of 0.77 g of the above compound as a viscous oil.

Example 78 Preparation of ##STR108##

The product of the previous example was saponified at room temperaturewith methanol/water/potassium hydroxide to give 6.41 g of the abovecompound as a white solid.

Example Preparation of ##STR109##

A 3 g (6.63 mmole, 1.0 equivalent) portion oft-butoxycarbonylnitroarginine-α-hydroxy-2-phenethylamide was taken up in15 mL of trifluoroacetic acid at 0° C. and stirred for 1.5 hours. Thissolution was concentrated twice with toluene in vacuo and taken up indimethylformamide. To this solution were added 1427 mg (6.63 mmole, 1.0equivalent) of t-butoxycarbonylproline acid, 7.3 mL (6705 mg, 66.3mmole, 10 equivalents) of NMM and 3225 mg (7.3 mmole, 1.1 equivalents)of BOP. Thin layer chromatography showed no reaction. More BOP and4-methylmorpholine were added and thin layer chromatography showed someproduct was forming. This solution was allowed to stir overnight,diluted with 1M HCl and extracted with ethyl acetate three times. Theorganics were combined and washed with 1M HCl, water (five times)saturated sodium bicarbonate, and brine; it was dried over magnesiumsulfate, filtered and reduced in vacuo to provide the above compound.

Example 80 Preparation of ##STR110##

A 550 mg (1.0 mmole, 1.0 equivalent) portion of the product of theprevious example was taken up in trifluoroacetic acid at 0° C. andstirred for 1.5 hours. This solution was concentrated twice with toluenein vacuo and taken up in dimethylformamide. To this solution were added265 mg (1.0 mmole, 1.0 equivalents) of the product of Example 78 and 487mg (1.1 mmole, 1.1 equivalents) of BOP. This solution was allowed tostir overnight and turned dark brown. This solution was diluted with 1NHCl and extracted three times with ethyl acetate. The organics werecombined, washed with 1N HCl, water (three times), saturated sodiumbicarbonate, and brine, and dried over magnesium sulfate, filtered, andreduced in vacuo to give 400 mg of the above compound as a white powder.

Example 81 Preparation of ##STR111##

A 200 mg (0.300 mmole, 1.0 equivalent) portion of the alcohol of theprevious example was taken up in 6 mL of 0.05M 1:1 DMSO:PheMe. To thissolution were added 577 mg (3.0 mmole, 10.0 equivalents) of EDC and0.099 mL (155 mg, 1.2 mmole, 4.0 equivalents) of dichloroacetic acid.This solution was allowed to stir for 1.5 hours, diluted with water andextracted two times with ethyl acetate. The organics were combined,washed with brine, dried over magnesium sulfate, and concentrated invacuo. This solution was purified using a 4:1:4hexanesmethanol:dichloromethane eluent on a silica column to give goodseparation. This gave 118 mg of the above compound that was stored inthe freezer under nitrogen.

Example 82 Preparation of ##STR112##

After hydrogen fluoride cleavage of the product of the previous examplethe molecular weight was found to be 649.4, in good agreement with theexpected value of 649.3.

Example 83 Preparation of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionicacid, methyl ester ##STR113##

2.0 g of 2-propylpentanoic acid is taken up in 10 mL of oxalyl chlorideand this mixture is stirred overnight at 23C. under nitrogen. After thistime, 100 mL of dry toluene is added and the volatiles removed in vacuoto yield the acid chloride which is used as indicated below.

1.0 g (2.5 mmole, 1 equiv.) of3-(N-3-benzyloxymethyl)tetrazolyl-2-(1,1-dimethylethoxy)methanamido-propionicacid, methyl ester is taken up in 10 mL of trifluoroacetic acid at -5°C. and this solution stirred for 0.5 hours followed by concentration invacuo.

The crude trifluoroacetate salt is taken up toluene and thisconcentrated again to remove any residual trifluoroacetic acid. Thecrude trifluoroacetate salt is then taken up in 5 mL of drytetrahydrofuran and 0.62 g (3.8 mmole, 1.5 equiv.) of 2-propylpentanoylchloride, prepared as indicated above, is added followed by the additionof 1.07 mL of triethylamine. The reaction mixture is stirred for 2 hoursat 23° C. and diluted with 50 mL of ethyl acetate. The organics arewashed with 0.5M HCl (2×25 mL), saturated sodium bicarbonate (25 mL),brine (25 mL), and dried over sodium sulfate. After decantation, theorganics are concentrated in vacuo and purified by chromatography onsilica (ethyl acetate/hexane eluent).

Example 84 Preparation of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionicacid ##STR114##

A 0.15 molar solution of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionicacid, methyl ester in methanol is prepared and 1.5 equivalents of a 1Mlithium hydroxide (aq.) is added. The reaction mixture is stirred untilno starting material remains by thin layer chromatography (about 3hours) and passed through Dowex 50x8-400 ion exchange resin and theresin washed with four column volumes of 1:1 methanol:water. Thefiltrate concentrated in vacuo to yield product in quantitative yield.

Example 85 Preparation of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionoyl-L-Prolyl-L-N^(g)-nitro-arginine, 2-phenethylketoamide ##STR115##

905 mg (2.25 mmole, 1 equiv.) of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionicacid, 1.56 g of L-prolyl-L-N^(g) -nitro-arginol, 2-phenethylketoamide,trifluoroacetate salt (2.25 mmole, 1 equiv.), 1.19 g of BOP reagent(2.25 mmole, 1 equiv.), and 34 mg of HOBt (0.2 mmole, 0.1 equiv.) werecombined in a 100 mL round bottom flask and 9 mL of dimethylformamidewas added followed by the addition of 1.48 mL (13.5 mmole, 6 equiv.) ofN-methylmorpholine. The reaction mixture was stirred at room temperaturefor 3 hours and then poured into a separatory funnel containing 100 mLof ethyl acetate and 10 mL of 3M HCl. The organics were washed with anadditional 10 mL of 0.5M HCl. The aqueous washes were combined and backextracted with 20 mL of ethyl acetate. The organics were combined andwashed with 10 mL of 1M sodium hydroxide followed by 10 mL of brine.After drying over sodium sulfate the organics were filtered andconcentrated in vacuo to yield 2.7 g of crude product.

This was taken up in 20 mL of DMSO and 20 mL of toluene. 4.32 g (22.5mmole, 10 equiv.) of EDC (water soluble carbodiimide) was added followedby the dropwise addition of 0.75 mL of dichloroacetic acid. This wasstirred at room temperature for 45 minutes. The solution was then pouredinto a separatory funnel containing 360 mL of ethyl acetate and 40 mL ofwater. The organics were washed with 50 mL of 0.5M HCl followed by 50 mLof brine. After drying over sodium sulfate the organics were filtered,concentrated in vacuo, and immediately chromatographed on silica (2%-10%methanol/methylene chloride gradient) to yield 1.59 g of product.

Example 86 Preparation of3-Tetrazolyl-2-(2-propylpentanoylamido)propionoyl-L-Prolyl-L-arginine,2-phenethylketoamide ##STR116##

The product of the previous example was cleaved with hydrogen fluorideand purified to give the above compound.

Example 87 Preparation of3-(N-2-Methyl)tetrazolyl-2-(2-propylpentanoylamido)propionic acid,methyl ester ##STR117##

2.0 g of 2-propylpentanoic acid is taken up in 10 mL of oxalyl chlorideand this mixture is stirred overnight at 23° C. under nitrogen. Afterthis time, 100 mL of dry toluene is added and the volatiles removed invacuo to yield the acid chloride which is used as indicated below. 1.0 g(2.5 mmole, 1 equiv.) of3-(N-3-benzyloxymethyl)tetrazolyl-2-(1,1-dimethylethoxy)methanamido-propionicacid, methyl ester is taken up in 10 mL of trifluoroacetic acid at -5°C. and this solution stirred for 0.5 hours followed by concentration invacuo. The crude trifluoroacetate salt is taken up toluene and thisconcentrated again to remove any residual trifluoroacetic acid.

The crude trifluoroacetate salt is then taken up in 5 mL of drytetrahydrofuran and 0.62 g (3.8 mmole, 1.5 equiv.) of 2-propylpentanoylchloride, prepared as indicated above, is added followed by the additionof 1.07 mL of triethylamine. The reaction mixture is stirred for 2 hoursat 23° C. and diluted with 50 mL of ethyl acetate. The organics arewashed with 0.5M HCl (2×25 mL), saturated sodium bicarbonate (25 mL),brine (25 mL), and dried over sodium sulfate. After decantation, theorganics are concentrated in vacuo and purified by chromatography onsilica (ethyl acetate/hexane eluent).

Example 88 Preparation of3-(N-2-methyl)tetrazolyl-2-(2-propylpentanoylamido)propionic acid##STR118##

A 0.15 molar solution of3-(N-2-benzyloxymethyl)tetrazolyl-2-(2-propylpentanoylamido)propionicacid, methyl ester in methanol is prepared and 1.5 equivalents of a 1Mlithium hydroxide (aq.) is added. The reaction mixture is stirred untilno starting material remains by thin layer chromatography (about 3hours) and passed through Dowex 50x8-400 ion exchange resin and theresin washed with four column volumes of 1:1 methanol:water. Thefiltrate concentrated in vacuo to yield product in quantitative yield.

Example 89 Preparation of3-(N-2-methyl)tetrazolyl-2-(2-propylpentanoyl-amido)propionoyl-L-Propyl-L-N^(g)-nitro-arginine,2-phenethylketoamide ##STR119##

410 mg (1.44 mmole, 1 equiv.) of3-(N-2-methyl)tetrazolyl-2-(2-propylpentanoylamido)propionic acid, 834mg (1.44 mmole, 1 equiv.) of L-prolyl-L-N^(g) -nitro-arginol,2-phenethylketoamide, trifluoroacetate salt, 636 mg (1.44 mmole, 1equiv.) of BOP, and 34 mg (0.14 mmole, 0.1 equiv.) of HOBt were combinedin a 100 mL round bottom flask and 7 mL of dimethylformamide was addedfollowed by the addition of 0.948 mL (8.63 mmole, 6 equiv.) ofN-methylmorpholine. The reaction mixture was stirred at room temperaturefor 3 hours and then poured into a separatory funnel containing 90 mL ofethyl acetate and 10 mL of 3M HCl. The organics were washed with anadditional 10 mL of 0.5M HCl. The aqueous washes were combined and backextracted with 20 mL of ethyl acetate. The organics were combined andwashed with 10 mL of 1M sodium hydroxide followed by 10 mL of brine.After drying over sodium sulfate the organics were filtered andconcentrated in vacuo to yield 1.67 g of crude product.

This was taken up in 20 mL of DMSO and 20 mL of toluene. 4.32 g (22.5mmole, 10 equiv.) of EDC (water soluble carbodiimide) was added followedby the dropwise addition of 0.75 mL of dichloroacetic acid. This wasstirred at room temperature for 45 minutes. The solution was then pouredinto a separatory funnel containing 360 mL of ethyl acetate and 40 mL ofwater. The organics were washed with 50 mL of 0.5M HCl followed by 50 mLof brine. After drying over sodium sulfate the organics were filtered,concentrated in vacuo, and immediately chromatographed on silica (2%-10%methanol/methylene chloride gradient) to yield 463 mg of product.

Example 90 Preparation of3-(N-2-methyl)tetrazolyl-2-(2-propylpentanoylamido)propionoyl-L-Prolyl-L-arginine,2-phenethylketoamide ##STR120##

The product of the previous example was cleaved with hydrogen fluorideto give the above compound.

The following compounds can also be made by those skilled in the artusing the methods of the present invention. ##STR121##

Example A Thrombin Assay

The ability of the compounds of the present invention to act asinhibitors of thrombin catalytic activity was assessed by determiningtheir inhibition constant, Ki, and the concentration which inhibitedenzyme activity by 50%, IC₅₀, against thrombin.

Enzyme activity was determined using the chromogenic substrates, S2266(H-D-valyl-L-leucyl-L-arginine-p-nitroaniline, obtained from KabiDiagnostica) or Pefachrome t-PA (CH₃ SO₂-D-hexahydrotyrosine-glycyl-L-Arginine-p-nitroaniline, obtained fromPentapharm Ltd.). The subtrates were reconstituted in deionized waterprior to use. Purified human α-thrombin was obtained from EnzymeResearch Laboratories, Inc. The buffer used for all assays was HBSA (10mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin).

The assay for Ki determinations was conducted by combining inappropriate wells of a Corning microtiter plate, 50 mL of HBSA, 50 mL ofthe test compound at a specified concentration diluted in HBSA (or HBSAalone for V_(o) (uninhibited velocity) measurement), and 50 mL of thechromogenic substrate S-2266 at a specified concentration diluted inHBSA. At time zero, 50 mL of α-thrombin diluted in HBSA, was added tothe wells yielding a final concentration of 0.5 nM in a total volume of200 mL. Velocities of S-2266 substrate hydrolysis which occurred over adesignated time period was measured by the change in absorbance at 405nm using a Thermo Max® Kinetic Microplate Reader.

Ki values were determined for test compounds using the followingmethodologies: 1) For test compounds exhibiting slow binding orslow-tight binding kinetics, Ki values were determined using therelationships developed by Williams and Morrison, Methods in Enzymology,63: 437 (1979) using steady state velocities (Vs) measured over 40minutes. The extent of substrate hydrolysis was less than 5% over thecourse of this assay. 2) For test compounds showing rapid, reversiblekinetics of inhibition, Ki values were determied from initial velocitiesusing the relationships developed by Dixon, M., Biochem. J., 129: 197(1972).

IC₅₀ determinations were conducted where HBSA (50 mL), α-thrombin (50μl) and inhibitor (50 μl) (covering a broad concentration range), werecombined in appropriate wells and incubated for 30 minutes at roomtemperature prior to the addition of substrate Pefachrome-t-PA (50 μl).The initial velocity of Pefachrome t-PA hydrolysis was measured by thechange in absorbance at 405 nm using a Thermo Max® Kinetic MicroplateReader over a 5 minute period in which less than 5% of the addedsubstrate was utilized. The concentration of added inhibitor whichcaused a 50% decrease in the initial rate of hydrolysis was defined asthe IC₅₀ value.

Table I below gives the Ki and IC₅₀ values for selected test compounds.The data shows their utility as potent in vitro inhibitors of humanα-thrombin.

                                      TABLE I    __________________________________________________________________________    Inhibitor Constants (Ki) and IC.sub.50 's of Compounds    Compound                                    Ki (nM)                                                     IC.sub.50    __________________________________________________________________________                                                     (nM)     ##STR122##                                 1.5  0.7     ##STR123##                                 5.5  6.5     ##STR124##                                 11.0 2.2     ##STR125##                                 --   0.42     ##STR126##                                 --   0.37     ##STR127##                                 0.09 0.36     ##STR128##                                 0.061                                                     0.48    __________________________________________________________________________

Example B Experimental Models of Thrombosis

The antithrombotic properties of the compound of Example 8 was evaluatedusing the following established experimental models of acute thrombosis.

Extracorporeal Shunt Model in Rats

This is one of the most common and generally used models in theevaluation of antithrombotic compounds. Smith, J. R. and White, A. M.Br. J. Pharmacol., 77: 29-38 (1982). In this model a localized clot madeup of primarily fibrin with some platelet and macrophage involvement(Shand, R. A. and Smith, J. R. and Wallis, R. B. Thromb. Res., 36:223-232 (1984)), is formed on an artificial thrombogenic surface(typically a segment of silk or cotton thread) contained in a sialsticchamber which is part of an exteriorized shunt between the carotidartery and jugular vein.

The effect of the compound of Example 8 on the formation of a thrombuson the thrombogenic surface was measured using clot weight as theprimary end point in the model.

Male Harlan Sprague Dawley rats (420-450 g) were acclimated at least 72hours prior to use. The animals were fasted for 12 hours prior tosurgery with free access to water. The animals were anesthetized with asodium pentobarbital (Nembutal) given intraperitoneally at a dose of 50mg/kg body weight and placed on a isothermal pad to maintain bodytemperature. The level of anesthesia was monitored every 15 minutes by:neuro-response to a tail pinch, respiration and core temperature. Thedesired depth of surgical anesthesia was maintained by administeringsubsequent doses (5 mg/kg) intravenously. The left femoral artery wascatheterized using standard procedures for blood pressure monitoring andblood sampling, with polyethylene tubing (PE50). The left and rightfemoral veins were catheterized with PE50 tubing for delivery ofanethestic and test compounds, respectively.

Following surgery the animals were randomized in either a control(saline infusion) or treatment group (Compound of Example 8) with atleast 6 animals per group per dose. The exteriorized shunt was assembledprior to catheterization by connecting two pieces of saline filled 12.5cm PE90 tubing with a 6 cm piece of PE160 tubing containing a 6 cm pieceof silk suture size 3 and clamped with hemostats. A small 0.5 cm portionof the silk thread protrudes from the junction of the chamber with theshunt. The left jugular vein and right carotid artery were catheterizedwith the ends of the PE90 shunt. Prior to unclamping the shunt, the testcompound (Compound of Example 8) was dissolved in normal saline, andinfused via the right femoral vein as an initial bolus (0.5 mg/kg)followed by a continuous intravenous infusion (at the designated dosesshown in the following table) for 30 minutes prior to exposure of thesuture to flowing blood. Blood pressure, heart rate core temperature andrespiration were monitored continuously. At the designated time, bloodflow through the chamber was initiated by unclamping the shunt andallowed to flow for a period of 15 minutes during which time the testcompound continued to be administered. At the end of the exposure periodboth sides of the chamber were clamped and the suture containing theclot removed following detachment of the arterial end of the chamber.The clot was immediately weighed and recorded. Following termination ofthe experiment the animal was euthanized with a 120 mg/kg dose ofNembutal. One experiment was performed per animal.

The efficacy of the compound of Example 8 as an antithrombotic agent inthis in vivo model was demonstrated by the reduction in clot size, asshown in Table II below.

                  TABLE II    ______________________________________    Efficacy of the Compound of Example 8 in Rat    Extracorporeal Shunt Model.    Treatment Group Clot size (mg).sup.a    ______________________________________    Control         41.30 ± 3.42    Group1          38.37 ± 4.49    Group2            .sup. 17.22 ± 1.79 *    Group3          .sup.   10.20 ± 0.636 *    ______________________________________     Control-no treatment     Group10.5 mg/kg i.v. bolus + 20 μg/kg/min i.v. infusion     Group20.5 mg/kg i.v. bolus + 50 μg/kg/min i.v. infusion     Group30.5 mg/kg i.v. bolus + 100 μg/kg/min i.v. infusion     α-weights are designated as the mean ± S.E.M. (n = 6).     * p ≦ 0.01 vs Control by oneway ANOVA followed by NewmanKuels Test

Rat model of FeCl₃ -induced platelet-dependent arterial thrombosis

This is a well characterized model of platelet dependent, arterialthrombosis which has been used in the evaluation potentialantithrombotic compounds such as direct thrombin inhibitors. Kurz, K.D., Main, B. W., and Sandusky, G. E., Thromb. Res., 60: 269-280 (1990).In contrast to the exteriorized shunt model, thrombus development inthis model is relatively heparin insensitive which suggests that thismodel may be more representative of the type of thrombosis which hasbeen observed clinically in newly re-canalized coronary vesselsfollowing balloon angioplasty or enzymatic thrombolysis. In this model aplatelet-rich, occlusive thrombus is formed in a segment of the ratcarotid artery treated with a fresh solution of FeCl₃ absorbed to apiece of filter paper. The FeCl₃ is thought to diffuse into the treatedsegment of artery and causes de-endothelialization resulting in thrombusformation. The effect of a test compound on the incidence of occlusivethrombus formation following the application of the FeCl₃ is monitoredby ultrasonic flowtometry and is used as the primary end point. The useof flowtometry is a modification of the original procedure in whichthermal detection of clot formation was employed. Kurz, K. D., Main, B.W., and Sandusky, G. E., Thromb. Res., 60: 269-280 (1990).

Male Harlan Sprague Dawley rats (420-450 g) were acclimated at least 72hours prior to use and fasted for 12 hours prior to surgery with freeaccess to water. The animals were prepared, anesthetized with Nembutalwith catheters for blood pressure monitoring, drug and anesthesiadelivery being implanted as described above. The left carotid artery wasisolated by making a midline cervical incision followed by bluntdissection and spreading techniques to separate a 2 cm segment of thevessel from the carotid sheath. A silk suture is inserted under theproximal and distal ends of the isolated vessel to provide clearance forthe placement of a ultrasonic flow probe (Transonic) around the proximalend of the vessel. The probe is then secured with a stationary arm.

Following surgery the animals were randomized in either a control(saline infusion) or treatment group with test compound (Compound ofExample 8) with at least 6 animals per group per dose. The testcompounds were administered as described above after placement of theflow probe and stabilization of the preparation for a period of 30 minprior to the thrombogenic stimulus. At t=0, a 3 mm diameter piece offilter paper (Whatman #3) soaked with 10 mL of a 35% solution of freshFeCl₃ (made up in water) was applied the segment of isolated carotidartery distal to the flow probe. Blood pressure, blood flow, heart rate,and respiration were monitored for 60 minutes.

The incidence of occlusion (defined as the attainment of zero bloodflow) was recorded as the primary end point. Following the 60 minuteobservation period the flow probe was removed and the area cleared ofall excess fluid. The distal and proximal sutures were tied off andarterial clamps placed on the far proximal and distal ends of thesegment. The isolated segment was cut out, blotted dry on filter paperand weighed. The segment was re-weighed following removal of the clotand the difference recorded as total % clot weight. The animals wereeuthanized as described above.

The efficacy of the compound of Example 8 as an antithrombotic agent inthis in vivo model was demonstrated by the reduction in the incidence ofocculsion and in clot size, as shown in Table III below.

                  TABLE III    ______________________________________    Results of the Compound of Example 8 in the FeCl.sub.3    Model of Thrombosis in Rats.    Treatment Group.sup.a                Incidence of Occlusion.sup.b                                Clot Size.sup.c    ______________________________________    Control     6/6             68.65 ± 3.75.sup.    Group1      5/6             40.73 ± 8.0 ***    Group2      .sup.  1/6 *    12.56 ± 5.96 ***    Group3      .sup.   0/6 **   4.46 ± 3.49 ***    ______________________________________     .sup.a -Controlno treatment     Group10.5 mg/kg i.v. bolus + 20 μg/kg/min i.v. infusion     Group20.5 mg/kg i.v. bolus + 50 μg/kg/min i.v. infusion     Group30.5 mg/kg i.v. bolus + 100 μg/kg/min i.v. infusion     .sup.b -Occlusion is defined as the establishment of zero blood flow     through the treated segment of the carotid artery.     .sup.c -Clot size is defined as: [Isolated clot/(Intact segmentEmpty     segment)] × 100. Numbers represent the mean ± S.E.M. (n = 6).     * p ≦ 0.05 vs Control by ChiSquare Analysis     ** p ≦ 0.005 vs Control by ChiSquare Analysis     *** p < 0.01 vs Control by oneway ANOVA followed by NewmanKuels Test

These in vivo data clearly demonstrated the antithrombotic efficacy ofthe Compound of Example 8 in two well established models of experimentalthrombosis.

We claim:
 1. A compound of the formula ##STR129## wherein (a) A₁ is R₁--S(O₂)--, wherein R₁ is selected from the group consisting ofalkyl of 1to about 12 carbon atoms, alkenyl of about 3 to about 6 carbon atoms,aryl of about 6 to about 14 carbon atoms which is optionallymono-substituted with X₁ or optionally di-substituted with X₁ and X₂,aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted in the aryl ring with X₁ or optionally di-substitutedin the aryl ring with X₁ and X₂, aralkenyl of about 8 to about 15 carbonatoms which is optionally mono-substituted in the aryl ring with X₁ oroptionally di-substituted in the aryl ring with X₁ and X₂,perfluoroalkyl of 1 to about 12 carbon atoms, perfluoroaryl of about 6to about 14 carbon atoms, trimethylsilylalkyl of 4 to about 8 carbonatoms, ##STR130## wherein X₁ and X₂ are independently selected from thegroup consisting of bromo, chloro, fluoro, Y₁ --, HO--, Y₁ --O--, NH₂--, Y₁ --NH--, (Y₁,Y₂)N--, Y₁ --C(O)--NH--, HS--, Y₁ --S--, Y₁ --S(O)--,Y₁ --S(O₂)--, HO--S(O₂)--, Y₁ --O--S(O₂)--, NH₂ --S(O₂)-- and Y₁--NH--S(O₂)--, wherein Y₁ and Y₂ are independently selected from thegroup consisting of trifluomethyl, pentafluoroethyl, alkyl of 1 to about12 carbon atoms, aryl of about 6 to about 14 carbon atoms, and aralkylof about 6 to about 15 carbon atoms; (b) A₂ is selected from the groupconsisting ofhydrogen, R₂ --, --(CH₂)_(m) --C(O)--O--H, --(CH₂)_(m)--C(O)--O--R₂, --(CH₂)_(m) --S(O₂)--R₂, --(CH₂)_(m) --S(O₂)--(CH₂)_(n)--C(O)--OH, --(CH₂)_(m) --S(O₂)--(CH₂)_(n) --C(O)--O--R₂, --(CH₂)_(m)--S(O₂)--O--H, --(CH₂)_(m) --S(O₂)--O--R₂, --(CH₂)_(m) --S(O₂)--NH₂,--(CH₂)_(m) --S(O₂)--NH--R₂, --(CH₂)_(m) --S(O₂)--NH--CH(R₃)--(CH₂)_(n)--C(O)--O--H, --(CH₂)_(m) --S(O₂)--NH--CH(R₃)--(CH₂)_(n) --C(O)--O--R₂,--(CH₂)_(m) --NH--S(O₂)--R₂, and --(CH₂)_(m) --NH--C(O)--O--R₂,wherein(i) m is 1, 2 or 3; (ii) n is 0, 1, 2, 3 or 4; (iii) R₂ is selected fromthe group consisting of alkyl of 1 to about 12 carbon atoms, alkenyl ofabout 3 to about 6 carbon atoms, aryl of about 6 to about 14 carbonatoms, aralkyl of about 6 to about 15 carbon atoms and aralkenyl ofabout 8 to about 15 carbons atoms; and (iv) R₃ is selected from thegroup consisting of hydrogen, alkyl of 1 to about 4 carbon atoms, arylof about 6 to about 14 carbon atoms, aralkyl of about 6 to about 15carbon atoms, and alkyl of 1 to about 4 carbon atoms substituted with asubstituent selected from the group consisting of --OH, --C(O)--OH,--C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃ and --NH--S(O₂)--CH₃ ;(c) A₃ is an amino acid residue of the amino acid selected from thegroup consisting of L-alanine, L-azetidinecarboxylic acid, glycine,L-isoleucine, L-leucine, L-lysine mono-substituted at its ε-amino groupwith R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine, L-ornithinemono-substituted at its δ-amino group with R₂ --S(O₂)--, L-pipecolicacid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline; and (d) A₄ is selected from the groupconsisting of ##STR131## wherein (i) p and q are each independentlyselected integers from 1 to 5 wherein the sum of p+q is 4 to 8;(ii) R₄is aryl of about 6 to about 14 carbon atoms which is optionallysubstituted with 1 or 2 substituents each independently selected fromthe group consisting of alkyl of 1 to about 4 carbon atoms, alkoxy of 1to about 4 carbon atoms, --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH,--NO₂ and --CF₃ ; (iii) R₅ is aryl of about 6 to 14 carbon atoms; (iv)R₆ is selected from the group consisting of hydrogen and alkyl of 1 toabout 4 carbon atoms; (v) R₇ is selected from the group consistingofhydrogen; alkyl of 1 to about 4 carbon atoms; aryl of about 6 to about14 carbon atoms which is optionally substituted with 1 or 2 substituentseach independently selected from the group consisting of --NH₂,--C(O)--OH, --C(O)--NH₂, fluoro, --OH, --NO₂, --CF₃, alkyl of 1 to about4 carbon atoms, and alkoxy of 1 to about 4 carbon atoms; aralkyl ofabout 6 to about 15 carbon atoms which is optionally substituted with 1or 2 substituents each independently selected from the group consistingof --NH₂, --C(O)--OH, --C(O)--NH₂, fluoro, --OH, --NO₂, --CF₃, alkyl of1 to about 4 carbon atoms, and alkoxy of 1 to about 4 carbon atoms; andalkyl of 1 to about 4 carbon atoms substituted with a substituentselected from the group consisting of --OH, --C(O)--OH, --C(O)--NH₂,--S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ; and (vi) R₈is selected from the group consisting of alkyl of 1 to about 12 carbonatoms, aryl of about 6 to about 14 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,and aralkyl of about 6 to about 15 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄,wherein X₃ and X₄ are independently selected from the group consistingof --C(O)--OH and --S(O₂)--OH, and(vii) r is 0, 1, 2 or 3; andpharmaceutically acceptable salts thereof.
 2. A compound of claim 1,wherein A₂ is --(CH₂)_(m) --C(O)--O--H.
 3. A compound of claim 2,wherein m is 1 or
 2. 4. A compound of claim 1, wherein A₂ is --(CH₂)_(m)--C(O)--O--R₂.
 5. A compound of claim 4, wherein R₂ is methyl.
 6. Acompound of claim 5, wherein m is
 1. 7. A compound of claim 1, whereinA₂ is --(CH₂)_(m) --S(O₂)--R₂.
 8. A compound of claim 7, wherein R₂ ismethyl.
 9. A compound of claim 8, wherein m is
 2. 10. A compound ofclaim 9, wherein A₁ is R₁ --S(O₂)--.
 11. A compound of claim 10, whereinR₁ is selected from the group consisting of alkyl of 1 to about 12carbon atoms, aryl of about 6 to about 14 carbon atoms and aralkyl ofabout 6 to about 15 carbon atoms.
 12. A compound of claim 11, wherein R₁is selected from the group consisting of 1-butyl, 2-naphthyl and benzyl.13. A compound of claim 12, wherein A₃ is selected from a groupconsisting of trans-4-hydroxy-L-proline, L-proline, L-leucine andL-isoleucine.
 14. A compound of claim 13, wherein A₃ is L-proline.
 15. Acompound of claim 14, wherein A₄ is selected from the group consistingof ##STR132##
 16. A compound of claim 15, wherein R₆ and R₇ are eachhydrogen.
 17. A compound of claim 16, wherein r is
 0. 18. A compound ofclaim 15, wherein A₄ is ##STR133##
 19. A compound of claim 18, whereinR₆ is hydrogen.
 20. A compound of claim 19, wherein R₇ is selected fromthe group consisting of alkyl of 1 to about 4 carbon atoms, aryl ofabout 6 to about 14 carbon atoms, aralkyl of about 6 to about 15 carbonatoms and alkylene of 1 to about 4 carbon atoms substituted with asubstituent selected from the group consisting of --OH, --C(O)--OH,--C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃.21. A compound of claim 20, wherein r is
 0. 22. A compound of claim 7selected from the group consisting of ##STR134##
 23. A compound of claim1, wherein A₁ is R₁ --S(O₂)--.
 24. A compound of claim 23, wherein R₁ isselected from the group consisting of alkyl of 1 to about 12 carbonatoms, aryl of about 6 to about 14 carbon atoms and aralkyl of about 6to about 15 carbon atoms.
 25. A compound of claim 24, wherein R₁ isselected from the group consisting of 1-butyl, 2-naphthyl and benzyl.26. A compound of claim 25, wherein A₃ is L-proline.
 27. A compound ofclaim 25, wherein A₄ is selected from the group consisting of ##STR135##28. A compound of claim 27, wherein R₆ and R₇ are each hydrogen.
 29. Acompound of claim 28, wherein r is
 0. 30. A compound of claim 27,wherein A₄ is ##STR136##
 31. A compound of claim 30, wherein R₆ ishydrogen.
 32. A compound of claim 31, wherein R₇ is selected from thegroup consisting of alkyl of 1 to about 4 carbon atoms, aryl of about 6to about 14 carbon atoms, aralkyl of about 6 to about 15 carbon atomsand alkylene of 1 to about 4 carbon atoms substituted with a substituentselected from the group consisting of --OH, --C(O)--OH, --C(O)--NH₂,--S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃.
 33. Acompound of claim 32, wherein r is
 0. 34. A compound of claim 1 selectedfrom the consisting of ##STR137##
 35. A compound of claim 26, wherein A4is ##STR138##
 36. A compound of claim 35, wherein R₆ and R₇ are eachhydrogen.
 37. A compound of claim 36, wherein r is
 1. 38. A compound ofclaim 37, wherein R₈ is selected from the group consisting of alkyl of 1to about 4 carbon atoms, aryl of about 6 to about 14 carbon atomsoptionally mono-substituted with X₃ or optionally di-substituted with X₃and X₄, and aralkyl of about 6 to about 15 carbon atoms optionallymono-substituted with X₃ or optionally di-substituted with X₃ and X₄.39. A compound of claim 38, wherein R₈ is selected from the groupconsisting of --CH(CH₃)₂, phenyl and benzyl.
 40. A pharmaceuticalcomposition comprising a pharmaceutically accceptable carrier and atherapeutically effective amount of the compound of claim
 1. 41. Amethod of preventing or reducing thrombotic occlusion in a mammalcomprising administering to said mammal a therapeutically effectiveamount of the compound of claim 1, 2, 4, 7, 10, 17, 21, 22, 23, 29, 33,34 or
 39. 42. A compound of the formula ##STR139## wherein (a) B₁ is R₉--S(O₂)--, wherein R₉ is selected from the group consisting ofalkyl of 1to about 12 carbon atoms, alkenyl of about 3 to about 6 carbon atoms,aryl of about 6 to about 14 carbon atoms which is optionallymono-substituted with X₅ or optionally di-substituted with X₅ and X₆,aralkyl of about 6 to about 15 carbon atoms which is optionallymono-substituted in the aryl ring with X₅ or optionally di-substitutedin the aryl ring with X₅ and X₆, aralkenyl of about 8 to about 15 carbonatoms which is optionally mono-substituted in the aryl ring with X₅ oroptionally di-substituted in the aryl ring with X₅ and X₆,perfluoroalkyl of 1 to about 12 carbon atoms, perfluoroaryl of about 6to about 14 carbon atoms, trimethylsilylalkyl of 4 to about 8 carbonatoms, ##STR140## wherein X₅ and X₆ are independently selected from thegroup consisting of bromo, chloro, fluoro, Y₃ --, Y₃ --O--, Y₃--O--C(O)--NH--, Y₃ --O--C(O)--N(Y₄)--, (Y₃,Y₄)N--, Y₃ --C(O)--NH--, Y₃--S--, Y₃ --S(O)--, Y₃ --S(O₂)--, Y₃ --O--S(O₂)--, NH₂ --S(O₂)-- and Y₃--NH--S(O₂)--, wherein Y₃ and Y₄ are independently selected from thegroup consisting of trifluoromethyl, pentafluoroethyl, aryl of about 6to about 14 carbon atoms, aralkyl of about 6 to about 15 carbon atomsand alkyl of 1 carbon atom to about 12 which is optionallymono-substituted with aralkyloxy of about 6 to about 15 carbon atoms;(b) B₂ is selected from the group consisting ofhydrogen, R₁₀ --,--(CH₂)_(s) --C(O)--O--R₁₀, --(CH₂)_(s) --S(O₂)--R₁₀, --(CH₂)_(s)--S(O₂)--(CH₂)_(t) --C(O)--O--R₁₀, --(CH₂)_(s) --S(O₂)--O--R₁₀,--(CH₂)_(s) --S(O₂)--NH--C(O)--O--R₁₀, --(CH₂)_(s) --S(O₂)--NH--R₁₀,--(CH₂)_(s) --S(O₂)--NH--CH(R₁₁)--(CH₂)_(t) --C(O)--O--R₁₀, --(CH₂)_(s)--NH--S(O₂)--R₁₀, and --(CH₂)_(s) --NH--C(O)--O--R₁₀,wherein (i) s is 1,2 or 3; (ii) t is 0, 1, 2, 3 or 4; (iii) R₁₀ is selected from the groupconsisting of alkenyl of about 3 to about 6 carbon atoms, aryl of about6 to about 14 carbon atoms, aralkyl of about 6 to about 15 carbon atoms,aralkenyl of about 8 to about 15 carbons atoms and alkyl of 1 to about12 carbon atoms which is optionally mono-substituted with aralkyloxy ofabout 6 to about 15 carbon atoms; and (iv) R₁₁ is selected from thegroup consisting of hydrogen, alkyl of 1 to about 4 carbon atoms, arylof about 6 to about 14 carbon atoms, aralkyl of about 6 to about 15carbon atoms, and alkyl of 1 to about 4 carbon atoms substituted with asubstituent selected from the group consisting of --O--R₁₀,--C(O)--O--R₁₀, --C(O)--NH₂, --S--CH₃, --S(O)--CH₃, --S(O₂)--CH₃ and--NH--S(O₂)--CH₃ ; (c) B₃ is an amino acid residue of the amino acidselected from the group consisting of L-alanine, L-azetidinecarboxylicacid, glycine, L-isoleucine, L-leucine, L-lysine mono-substituted at itsε-amino group with R₂ --S(O₂)--, L-methionine sulfone, N-methylglycine,L-ornithine mono-substituted at its δ-amino group with R₂ --S(O₂)--,L-pipecolic acid, L-phenylalanine, L-proline, L-valine, andtrans-4-hydroxy-L-proline substituted at 4-hydroxy group with R₁₂--O--C(O)--, wherein R₁₂ is selected from the group consisting of alkylof 1 to about 12 carbon atoms and aralkyl of about 6 to about 15 carbonatoms; and (d) B₄ is selected from the group consisting of ##STR141##wherein (i) u and v are each independently selected integers from 1 to 5wherein the sum of u+v is 4 to 8;(ii) R₁₃ is aryl of about 6 to about 14carbon atoms which is optionally substituted with 1 or 2 substituentseach independently selected from the group consisting of alkyl of 1 toabout 4 carbon atoms, alkoxy of 1 to about 4 carbon atoms,--NH--C(O)--O--X₇, --C(O)--O--X₇, --C(O)--NH₂, fluoro, --O--X₇, --NO₂and --CF₃ ; (iii) R₁₄ is aryl of about 6 to about 14 carbon atoms; (iv)R₁₅ is selected from the group consisting of hydrogen and alkyl of 1 toabout 4 carbon atoms; (v) R₁₆ is selected from the group consistingofhydrogen, alkyl of 1 to about 4 carbon atoms, aryl of about 6 to about14 carbon atoms which is optionally substituted with 1 or 2 substituentseach independently selected from the group consisting of--NH--C(O)--O--X₈, --C(O)--O--X₈, --C(O)--NH₂, fluoro, --O--X₈, --NO₂,CF₃, alkyl of 1 to about 4 carbon atoms, and alkoxy of 1 to about 4carbon atoms, aralkyl of about 6 to about 15 carbon atoms which isoptionally substituted with 1 or 2 substituents each independentlyselected from the group consisting of --NH--C(O)--O--X₉, --C(O)--O--X₉,--C(O)--NH₂, fluoro, --O--X₉, --NO₂, --CF₃, alkyl of 1 to about 4 carbonatoms, and alkoxy of 1 to about 4 carbon atoms, and alkyl of 1 to about4 carbon atoms substituted with a substituent selected from the groupconsisting of --O--X₁₀, --C(O)--O--X₁₀, --C(O)--NH₂, --S--CH₃,--S(O)--CH₃, --S(O₂)--CH₃, and --NH--S(O₂)--CH₃ ; (vi) R₁₇ is selectedfrom the group consisting of alkyl of 1 to about 4 carbon atoms andaralkyl of about 6 to 15 carbon atoms; (vii) R₁₉ is selected from thegroup consisting of hydrogen atom, aryl of about 6 to about 14 carbonatoms which is optionally mono-substituted with X₁₁ or optionallydi-substituted with X₁₁ and X₁₂ ; and aralkyl of about 6 to about 15carbon atoms which is optionally mono-substituted with X₁₁ or optionallydi-substituted with X₁₁ and X₁₂ ; and (viii) w is 0, 1, 2, 3, 4 or5;wherein X₇, X₈, X₉ and X₁₀ are independently selected from the groupconsisting of alkyl of 1 to about 4 carbon atoms, aryl of about 6 toabout 14 carbon atoms and aralkyl of about 6 to about 15 carbon atoms;and wherein X₁₁ and X₁₂ are independently selected from the groupconsisting of --C(O)--O--R₁₇ and --S(O₂)--O--R₁₇.
 43. A compound ofclaim 42, wherein B₂ is --(CH₂)_(s) --C(O)--O--R₁₀.
 44. A compound ofclaim 43, wherein R₁₀ is selected from the group consisting of benzyland benzyloxymethyl.
 45. A compound of claim 44, wherein s is 1 or 2.46. A compound of claim 43, wherein R₁₀ is alkyl of 1 to about 12 carbonatoms.
 47. A compound of claim 46, wherein R₁₀ is methyl.
 48. A compoundof claim 42, wherein B₂ is --(CH₂)_(s) --S(O₂)--R₁₀.
 49. A compound ofclaim 48, wherein R₁₀ is methyl.
 50. A compound of claim 49, wherein sis
 2. 51. A compound of claim 50, wherein B₁ is R₉ --S(O₂)--.
 52. Acompound of claim 51, wherein R₉ is selected from the group consistingof 1-butyl, 2-naphthyl and benzyl.
 53. A compound of claim 52, whereinB₃ is selected from the group consisting of L-proline, L-leucine,L-isoleucine and trans-4-hydroxy-L-proline substituted at 4-hydroxygroup with benzyloxycarbonyl.
 54. A compound of claim 53, wherein B₄ isselected from the group consisting of ##STR142##
 55. A compound of claim54, wherein R₁₅ and R₁₆ are each hydrogen.
 56. A compound of claim 55,wherein w is
 0. 57. A compound of claim 56, wherein R₁₇ is benzyl andR₁₈ is benzyloxymethyl.
 58. A compound of claim 42, wherein B₂ ishydrogen.
 59. A compound of claim 58, wherein B₁ is R₉ --S(O₂)--.
 60. Acompound of claim 59, wherein R₉ is selected from the group consistingof 1-butyl, 2-naphthyl and benzyl.
 61. A compound of claim 60, whereinB3 is L-proline.
 62. A compound of claim 61, wherein B₄ is selected fromthe group consisting of ##STR143##
 63. A compound of claim 62, whereinR₁₅ and R₁₆ are each hydrogen.
 64. A compound of claim 63, wherein w is0.
 65. A compound of claim 64, wherein R₁₇ is benzyl.
 66. A compound ofclaim 61, wherein B₄ is ##STR144##
 67. A compound of claim 66, whereinR₁₅ and R₁₆ are each hydrogen.
 68. A compound of claim 67, wherein wis
 1. 69. A compound of claim 68, wherein R₁₉ is selected from the groupconsisting of --CH(CH₃)₂, phenyl and benzyl.
 70. A compound of claim 1,selected from the group consisting of: ##STR145##
 71. A compound ofclaim 1 having the formula: ##STR146##
 72. A compound of claim 1 havingthe formula: ##STR147##
 73. A compound of claim 1 having the formula:##STR148##